U.S. patent application number 16/816221 was filed with the patent office on 2020-09-17 for nanopore sensing device, components and method of operation.
This patent application is currently assigned to Oxford Nanopore Technologies Inc.. The applicant listed for this patent is Oxford Nanopore Technologies Inc.. Invention is credited to Ken Healy, Justin Millis, Ping Xie.
Application Number | 20200292521 16/816221 |
Document ID | / |
Family ID | 1000004854676 |
Filed Date | 2020-09-17 |
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United States Patent
Application |
20200292521 |
Kind Code |
A1 |
Xie; Ping ; et al. |
September 17, 2020 |
NANOPORE SENSING DEVICE, COMPONENTS AND METHOD OF OPERATION
Abstract
Devices for improved nanopore sensing are described. An example
device has a structure arranged to separate an analyte reservoir
and an outlet chamber. An example device has a structure arranged
to separate an analyte reservoir and an outlet chamber. The
structure can include an array of nanopore structures, each
nanopore structure comprising a passage for fluid connection
through the structure between the analyte reservoir and outlet
chamber. Control terminals can be arranged for applying a control
signal to alter the electrical potential difference across that
nanopore structure. Some embodiments include an electronic circuit
configured to detect a signal from an electrical transduction
element at each nanopore structure. Additional structural features
and methods of operating and making the devices are described.
Inventors: |
Xie; Ping; (Cambridge,
MA) ; Millis; Justin; (Cambridge, MA) ; Healy;
Ken; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Oxford Nanopore Technologies Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
Oxford Nanopore Technologies
Inc.
Cambridge
MA
|
Family ID: |
1000004854676 |
Appl. No.: |
16/816221 |
Filed: |
March 11, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62817211 |
Mar 12, 2019 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 27/128 20130101;
G01N 33/48721 20130101 |
International
Class: |
G01N 33/487 20060101
G01N033/487; G01N 27/12 20060101 G01N027/12 |
Claims
1. A device for nanopore sensing, said device comprising: a
structure arranged to separate an analyte reservoir and an outlet
chamber, the structure comprising an array of nanopore structures,
one or more of the nanopore structures comprising a passage for
fluid connection through the structure between the analyte
reservoir and outlet chamber; drive electrodes connected
respectively in the analyte reservoir and the outlet chamber for
imposing an electrical potential difference across the passage;
electrical transduction elements, each element connected to the
passage of a respective nanopore structure for measuring the
fluidic electrical potential at that electrical transduction
element in that nanopore structure; and control terminals, each
terminal connected to a respective nanopore structure for applying
a control signal to alter the electrical potential difference
across that nanopore structure.
2. A device according to claim 1, wherein the electrical
transduction element and the control terminal associated with each
nanopore structure are directly connected.
3. A device according to claim 1, wherein the terminals are
configured to apply a control signal to alter the electrical
potential difference across each respective nanopore structure in
response to a measurement of the fluidic electrical potential at
the electrical transduction element at that nanopore structure.
4. A device according to claim 3, wherein the application of the
control signal is configured to alter the potential difference
between at least one of the control terminals and at least one of
the drive electrodes.
5. A device according to claim 3, wherein the control signal is
connectable to a plurality of the nanopore structures to
simultaneously alter the potential difference between the connected
control terminals and at least one of the drive electrodes.
6. A device according to claim 1, wherein the electrical
transduction elements are isolatable from a measuring circuit.
7. A device according to claim 3, wherein the electrical
transduction elements are isolatable prior to the application of
the control signal.
8. A device according to claim 1, wherein a nanopore structure
comprises a nanopore.
9. A device according to claim 8, wherein the control signal is
applied for the purpose of altering the potential difference across
the nanopore in order to: unblock the passage of a nanopore when
the device detects that an analyte is blocked; reject an analyte
being measured; and/or alter the direction and/or speed of
translocation of an analyte through the nanopore an analyte.
10. A device according to claim 1, wherein the array has electronic
circuits, each electronic circuit associated with at least one
respective nanopore structure and connected to the electrical
transduction element, each electronic circuit configured to modify
and/or process the signals received therefrom.
11. A device according to claim 10, wherein each electronic circuit
is associated with a group of nanopore structures.
12. A device according to any preceding claim, wherein the array
has control circuits, each control circuit associated with a
respective nanopore structure and connected to the control terminal
and/or the electrical transduction element, the control circuit
configured to alter at one or more of the respective nanopore
structures an electrical potential imposed by the drive electrodes
in response to a signal.
13. A device according to claim 12, wherein each control circuit is
associated with a group of nanopore structures.
14. A device according to claim 1, wherein the structure has: a
nanopore layer incorporating a nanopore and/or incorporating a well
for supporting a nanopore; and a base layer incorporating channels,
wherein the nanopore layer and the base layer are sandwiched
together such that the nanopore nanopores and/or wells are aligned
to define the passage.
15. A device according to claim 13, wherein at least one of the
electrical transduction element, the control circuit, or the
control terminal are disposed on or below the outer surface of the
structure.
16. A device having nanopore structures for sensing an analyte, the
nanopore structures configured in a structure, said structure
arranged to separate an analyte reservoir and an outlet chamber,
each nanopore structure providing a passage for fluid connection
through the structure between the analyte reservoir and outlet
chamber, wherein each nanopore structure comprises: an electrical
transduction element; and an electronic circuit configured to
detect a signal from the electrical transduction element, wherein
each of the electronic circuits are configured to perform one of,
or some combination of, store, transmit, process and communicate at
least a portion of the signal to a connectable processor.
17. (canceled)
18. A device according to claim 16, wherein each of the nanopore
structures in the structure further comprise a compensation
circuit.
19. A device according to claim 18, wherein the compensation
circuit has a variable gain amplifier and/or a variable capacitor
in a feedback loop of the compensation circuit.
20. A device according to claim 16, wherein each of the nanopore
structures have a control terminal, each control terminal
associated with a respective nanopore for applying a control signal
to alter the electrical potential difference across that
nanopore.
21. A device according to claim 20, wherein the control terminal is
switchably connected to a power supply to change the configurable
voltage level imposed upon the pore.
22-96. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn. 119(e) to U.S. Provisional Patent Application Ser.
No. 62/817,211, filed Mar. 12, 2019, and entitled "NANOPORE SENSING
DEVICE, COMPONENTS AND METHOD OF OPERATION," which is incorporated
herein by reference in its entirety for all purposes.
FIELD
[0002] The described technology relates to a device for nanopore
sensing, having a plurality (e.g., an array) of nanopore structures
configurable as nanopore sensors, as well as methods for operating
nanopore sensors and/or for fabricating an array of nanopore
structures.
BACKGROUND
[0003] Nanopore sensors have been developed for sensing a wide
range of species, including single molecules such as polymer
molecules. An example of a nanopore sensor device is a MinION.TM.,
manufactured and sold by Oxford Nanopore Technologies Ltd. The
nanopore-based sensing therein employs the measurement of ionic
current flow through a biological nanopore located in a highly
resistive amphiphilic membrane. The MinION.TM. has an array of
nanopore sensors. As a molecule, such as a polymer analyte (e.g.
DNA), is caused to translocate a nanopore, measurement of the
fluctuations in ionic current may be used to determine the sequence
of the DNA strand. Nanopore devices for detection of analytes other
than polynucleotides such as proteins are described in
WO2013/123379 published on 22 Aug. 2013.
[0004] An alternative to biological nanopore devices, such as
MinION.TM., are solid state nanopore devices. FIG. 1 shows a
portion of a single sensor device 2 with a solid-state nanopore 4
disclosed in WO2016/127007, published on 11 Aug. 2016, hereby
incorporated by reference in its entirety, in which an analyte 6
passes through a body 8 from a cis reservoir 10, through the
solid-state nanopore 4 and into a fluidic passage 12; a signal is
read via a sensor 16 located close to the nanopore. Electrodes 18
are provided in the reservoirs 10, 14 for inducing the analyte
through the nanopore.
SUMMARY
[0005] The performance of solid-state nanopore sensors is limited
by the sensing components, manufacturing techniques and their
tolerances, which can occur as a result of variation in the
formation of the nanopore or the assembly of the sensor. These and
other factors detriment the bandwidth, sensitivity and ability to
control such nanopore sensors.
[0006] An aspect of the described embodiments is to overcome
problems associated with implementing a nanopore sensor array
having a plurality of nanopore sensors.
[0007] The present inventors sought to improve upon nanopore
sensing devices by providing, in some aspects, the ability to
control the movement of an analyte while also improving the
measurement accuracy by mitigating factors that impede on the
measurement, such as noise caused by parasitics and contaminated
sensing components. Moreover, the improved devices allow the
nanopore structures, and nanopore sensors implemented therefrom, to
be formed in large arrays in an efficient manner without inhibiting
the control or performance of the array.
[0008] In a first aspect, some embodiments include a device for
nanopore sensing, said device having: a structure arranged to
separate an analyte reservoir and an outlet chamber, the structure
comprising an array of nanopore structures, each nanopore structure
comprising a passage for fluid connection through the structure
between the analyte reservoir and outlet chamber;
[0009] drive electrodes connected respectively to the analyte
reservoir and the outlet chamber for imposing an electrical
potential difference across the passages;
[0010] electrical transduction elements, each element connected to,
or exposed to, the passage of a respective nanopore structure for
measuring the fluidic electrical potential at that electrical
transduction element in that nanopore structure; and
[0011] control terminals, each terminal connected to a respective
nanopore structure for applying a control signal to alter the
electrical potential difference across that nanopore structure or
to alter an electrical potential within the passage.
[0012] The structure can be a support structure. The nanopore
structures can be disposed in and/or on the corresponding array of
passages. Each nanopore structure can have an aperture forming part
of the passage. Each nanopore structure in the array of nanopore
structures has a respective passage. The control terminal can be
connected to a respective passage in that structure for applying a
control signal to alter the fluidic electrical potential
distribution around a respective nanopore structure. When provided
with a fluid, such that there is a fluid connection between the
drive electrodes and the nanopore structure, then the control
signal applied to that nanopore structure can alter the electrical
potential difference across that nanopore structure with respect to
the drive electrodes. The control terminal can be connected to the
electrical transduction element. The control terminal can be
switchably connected to the electrical transduction element.
[0013] The nanopore structures of the array can have a nanopore, be
capable of supporting a nanopore or be capable of supporting a
membrane having a nanopore.
[0014] In operation as a nanopore sensing device, the device
comprises an array of nanopores.
[0015] When provided with a fluid, fluidic electrical potential can
be measured at the electrical transduction element. When provided
with a fluid, the fluidic electrical distribution around that
nanopore structure can be altered.
[0016] In operation, fluid resides in the analyte reservoir, outlet
chamber and passages of the device wherein the reservoir and
chamber are fluidically connected. The fluid in the reservoir,
chamber and passages of the nanopore structure can be different
fluids.
[0017] A nanopore structure may comprise an aperture having a width
of nanometer dimensions. It may be a through hole in a solid-state
support, such as a solid state nanopore.
[0018] Alternatively, in an embodiment, the nanopore structure may
be a structure that is capable of supporting a nanopore to provide
a passage of nanometer dimensions. In this embodiment the nanopore
structure may comprise an aperture of micrometre or nanopore
dimensions. Exemplary nanopore structures that may be used to
support a nanopore are disclosed in WO2014/064443, hereby
incorporated by reference in its entirety. Examples of nanopores
that may be supported by the nanopore structure are biological
nanopores such as protein nanopores. The nanopore may be provided
in a membrane such as an amphiphilic membrane. The membrane may be
supported by the nanopore structure.
[0019] In some embodiments, when used for nanopore sensing, the
device may comprise (e.g., comprises) an array of nanopores.
[0020] The analyte reservoir can function to receive an analyte for
sensing by the nanopore array. The outlet chamber can function to
receive an analyte that passes through the nanopore array.
[0021] The nanopores (where present) separate a cis side and a
trans side of the device. The analyte reservoir may be considered
as the cis side of the device and the analyte outlet chamber may be
considered as being part of the trans side.
[0022] The device may be provided with or without fluid. The fluid
in the reservoir, chamber and passages of the nanopore structure
can be different fluids.
[0023] Some embodiments relate to a structure comprising an array
of nanopore structures, each nanopore structure comprising a
passage for fluid connection through the structure. Each nanopore
structure has an electrical transduction element, each element
connected to, or exposed to, the passage of a respective nanopore
structure for measuring the fluidic electrical potential at that
electrical transduction element in that nanopore structure. Each
nanopore structure also has a control terminal, each terminal
connected to a respective nanopore structure for applying a control
signal to alter the fluidic electrical potential distribution
within the passage or around a respective nanopore structure.
[0024] The structure can be a support structure. The nanopore
structures can be disposed in and/or on the corresponding array of
passages. Each nanopore structure can have an aperture forming part
of the passage. Each nanopore structure in the array of nanopore
structures has a respective passage. The control terminal can be
connected to a respective passage in that structure for applying a
control signal to alter the fluidic electrical potential
distribution around a respective nanopore structure. Each aperture
of the array can be associated with a respective electrical
transduction element and a control terminal.
[0025] Each nanopore structure in the array of nanopore structures
can be considered a pixel, each pixel comprising an aperture, an
electrical transduction element and a control terminal. An array of
pixels can be arranged as a rectilinear grid in a manner analogous
to the arrangement of pixels on a television screen. The nanopores
when present in the nanopore structure forms part of the passage,
namely a section of the passage is of nanometer width. The nanopore
may be a solid state nanopore, namely wherein an aperture of
nanopore width is provided in a solid support. Alternatively, the
nanopore may be a hybrid nanopore, wherein a biological nanopore is
provided in an aperture of a solid support. The biological nanopore
may be supported in an amphipathic membrane. The amphipathic
membrane may be supported by pillars such as disclosed in
WO2014/064443. The nanopore structure capable of supporting a
nanopore may comprise an aperture of a width greater than nanopore
dimensions, such as micrometre dimensions. The nanopore structure
may comprise means by which to support an amphipathic membrane. The
cis can be used to store an analyte, such as an analyte, for
analysis. The analyte can be passed through a nanopore in a
nanopore sensor of the array. After passing through the nanopore
the analyte can either remain in the passage or pass out of the
passage in to the outlet chamber. When the cis, trans and passages
of the array of nanopore structures are provided with a fluid the
drive electrodes can impose an electrical potential difference
across the passage. The drive electrodes can provide a potential
difference across the apertures to induce passage of a charged
analyte through a nanopore of the array. The potential difference
can be altered to change the speed or direction of translocation of
the analyte.
[0026] Each electrical transduction element in the array functions
as a sensor electrode. Changes in ion current flow through the
nanopore cause fluctuations in electrical potential caused by
changes in ion current flow, said electrical potential may be
measured to determine the presence or a property of an analyte. The
fluid in the device, which can be aqueous, may contain ions.
Multiple analytes may be translocated
[0027] The drive electrode serves to provide a common potential
difference across the array of nanopores, wherein multiple analytes
may be measured simultaneously in the array. Measurements are made
at the electrical transduction elements in each nanopore
structure.
[0028] In some embodiments, each nanopore structure may have an
associated control terminal. This terminal can be an independent
connection to a control signal generated externally from the
structure. This allows the electrical potential to be applied
independently of altering electrical potential differences across
other nanopore structures in the array. The control signal can be
generated within the nanopore structure in response to an external
trigger or switch. Or, the control signal can be generated from a
circuit internal to that nanopore structure. The control signal has
the effect of changing the voltage level at each nanopore
structure. The control signal can be applied via the electrical
transduction element for modifying the voltage between the passage
and the drive electrode(s). Additionally, or alternatively, the
control signal can be applied via an electrical connection, such as
a terminal or further control electrode, in the passage.
[0029] The device may have a single drive electrode provided in
electrical connection with the analyte reservoir and a single drive
electrode provided in electrical connection with the outlet chamber
wherein the drive electrode serves to provide a common potential
difference across the nanopore array.
[0030] Alternatively, the device may comprise a plurality of drive
electrodes on the cis and/or the trans side of the device.
[0031] The application of a control signal to an individual
nanopore structure can function to change the potential difference
across the nanopore structure, i.e. between that nanopore structure
and the drive electrodes. By way of example, the drive electrode in
the cis can have a voltage level of -0.1 volts, while the drive
electrode in the trans can have a voltage level of 0.2 volts such
that the potential difference across the passages of the array is
0.3 volts. The application of a control signal to impose a voltage
of -2 volts at the nanopore structure results in a potential
difference between the nanopore structure and the cis and trans
electrodes of -1.9 volts and -2.2 volts, respectively, or a
potential difference between the nanopore structure and the cis and
trans electrodes of -1.9 volts and -1.8 volts, respectively.
[0032] The electrical transduction element and the control terminal
of each nanopore structure can be directly connected. In doing so,
the electrical transduction element can function as both a sensor
electrode and a control electrode. This can be implemented by
providing an electrical transduction element with two terminals:
one for connecting to sensing circuitry, the other for connecting
to control circuitry. In practice, the sensing circuitry and the
control circuitry can reside in the same circuit or component. Any
of the circuits can be located off-structure and connected to the
structure via, for example, a wire-bond.
[0033] The control terminals can be configured to apply a control
signal to alter the electrical potential difference from the drive
electrodes to each respective nanopore structure in response to a
measurement of the fluidic electrical potential at the electrical
transduction element of that nanopore structure by said electrical
transduction element. The application of the control signal can be
configured to alter the potential difference between at least one
of the control terminals and at least one of the drive
electrodes.
[0034] A control signal applied to the control terminal of a
nanopore structure can alter the magnitude and/or the polarity of
the potential difference between that nanopore structure and a
drive electrode, which can change the rate at which an analyte
passes though the passage of that nanopore structure or change the
direction of movement of that analyte.
[0035] The control signal can be connectable to a plurality of the
nanopore structures to simultaneously alter the potential
difference between the connected control terminals and at least one
of the drive electrodes.
[0036] The control signal can be applied for purposes other than to
reject an analyte or control the speed and or direction of its
translocation. For example, the control signal can be applied to
induce insertion of a biological nanopore in a membrane supported
by the nanopore structure. The electrical transduction elements can
be connected to a measuring circuit to read signals received from
the electrical transduction element. The nanopore structure can be
provided with a switchable connection to a measurement circuit.
Said switchable connection can disconnect the measurement circuit
prior to the application of a control signal. In this way the
control signal can be disconnected from measurement circuitry and
inhibit the control signal influencing the performance of
measurement circuitry.
[0037] In other words, the electrical transduction elements can be
isolatable prior to the application of the control signal. Each
individual electrical transduction element of each nanopore
structure can be selectively isolated prior to application of the
control signal.
[0038] The control signal can be applied for various purposes.
[0039] The control signal can be applied independently of
measurements of the analyte. For example, the control signal can be
applied to a membrane supported by the nanopore structure to induce
insertion of a biological nanopore in the membrane.
[0040] The control signal can be applied to a nanopore structure in
response to a measurement by the electrical transduction
element.
[0041] By way of example, the control signal can be applied to for
the purpose of unblocking a nanopore when the device determines
that the passage through the nanopore is blocked, for example by
analyte. The control signal can then be applied to unblock the
passage.
[0042] The device is able to determine that the nanopore is blocked
from the measurement of the change in electrical potential caused
by the inhibition of current flow through the nanopore. In the
absence of interaction of analyte with the nanopore, ion current
flow through the nanopore due to the presence of an ionic salt in
the aqueous sample may be referred to as the open pore current.
When an analyte interacts with the nanopore, ion current flow
through the pore is reduced and variation in the reduction in ion
current may be measured as a fluctuation in electrical potential at
the sensor electrode (e.g., sensor electrode 126) over time as an
analyte such as DNA translocates the nanopore. A blockage of the
nanopore, for example due to analyte becoming immobilised in the
pore gives rise to a reduced ion current flow whose value changes
very little over time. In a further example, the control signal can
be applied to eject an analyte from the nanopore which is not of
interest or which is no longer of interest. Measurements can be
performed in real-time such that a decision to eject the analyte
may be made before complete measurement of the analyte, for example
a polynucleotide is made.
[0043] With regard to the prior mentioned devices for sequencing
polynucleotides such as the MinION.TM. device, current flow though
the nanopores is measured under the application of a potential
difference between a respective array of electrodes provided on one
side of each the nanopores and a common electrode provided on the
other side of the electrodes in an analyte reservoir. Because each
nanopore has an associated electrode, it is possible to
individually control the potential difference across each nanopore
of the array and eject an analyte. In the hereinafter described
embodiments, there are various advantages associated with carrying
out measurement of the local potential at each nanopore by means of
the electrical transduction elements. The drive electrodes serve to
provide a potential difference across the nanopore array and not to
measure analyte. Consequently, individual control of the potential
difference at a nanopore by the drive electrodes is not possible.
However, it is possible to provide individual control over the
potential difference across each nanopore by means control
terminals.
[0044] The array of nanopore structures can have circuits, each
circuit associated with a respective nanopore structure and
connected to the electrical transduction element. Each circuit can
be configured to modify and/or process the signals received from
the electrical transduction element. The circuit can also apply a
control signal to the electrical transduction element. The circuit
can isolate the control signal applied to the electrical
transduction element from other sensing and processing
functions.
[0045] Each circuit can reside within the pixel of the nanopore
structure. Each circuit can be addressable. Each nanopore structure
can be addressable. The addressing function can allow an external
processor to communicate with a nanopore structure to at least one
of receive measurement information or control movement of an
analyte in the passage. In this way, the measurement and control of
sensing at each individual passage can be independently controlled.
The circuits may be provided on or embedded within the support
structure.
[0046] Each electronic circuit can be associated with a group of
nanopore structures. By way of example an electronic circuit can be
shared by a group of four nanopore structures. Sensing and control
of the nanopore structures in the group can be multiplexed. In this
way the circuit can be addressable, and multiplexing used to
control individual nanopore structures.
[0047] Each circuit may be associated with a respective nanopore
structure or a group of nanopore structures. Each circuit can be
connected to the control terminal and/or the electrical
transduction element, such that the circuit configured to alter at
the respective nanopore structure an electrical potential imposed
by the drive electrodes in response to a measurement at the
electrical transduction element and/or from an external processor
attached thereto.
[0048] The structure can have a nanopore layer incorporating a
nanopore and/or incorporating a well for supporting a solid-state
film or a membrane having a nanopore. When provided with a nanopore
the nanopore structure can be operated as a nanopore sensor. The
nanopore layer can be provided with nanopore after the nanopore
structure has been made. Nanopores can be provided by a user after
a device having nanopore structures has been provided to them. The
nanopore layer can be replaced such that the device is recyclable.
The nanopore structure can also include a base layer incorporating
channels. The nanopore layer and the base layer can be sandwiched
or laminated together such that the nanopores and/or wells are
aligned to define the passage. At least one of the electrical
transduction element, the circuit, or the control terminal are
disposed on or between the outer surface of the structure. The
individual nanopore structures may be comprised of a single
structure or one or more sub-structures connected to each other.
The single or sub-structures may be planar or sheet like.
[0049] Each nanopore structure can be defined by its passage. The
passage can fluidly connect a cis and trans. The passage can be
formed by formations in each nanopore structure which, by way of
example, is formed by: a nanopore layer for supporting a nanopore,
the layer having a through-hole; a base layer having a channel,
which functions as a through-hole. The through-holes of the
nanopore layer and the base layer are aligned to for form the
passage.
[0050] The electrical transduction element defines a part of the
passage. By way of example, the electrical transduction element can
be sandwiched or laminated between the nanopore layer and the base
layer. It can, however, be located elsewhere in the passage. It can
be configured around the passage provided there is a fluid
connection, and can be a direct fluid connection, between the
electrical transduction element and a nanopore provided in the
nanopore layer.
[0051] The electrical transduction element and/or the circuit can
be implemented on a sense layer. The sense layer can be a
sub-structure. The sense layer can be sandwiched or embedded
between the nanopore layer and the base layer, said sense layer
having a through-hole that aligns with the through-holes of the
nanopore layer and the base layer. To be clear, the nanopore layer,
sense layer and base layer can be sub-structures that are stacked
to provide an array of nanopore structures.
[0052] A nanopore, when provided in the nanopore structure, forms
part of the passage. The rejection of an analyte can be managed
using a control signal, which functions to control the movement of
an analyte in the nanopore, e.g. reject the analyte from the
nanopore. The nanopore in a passage can become blocked. The
blockage of a nanopore can be sensed and a control signal applied
to the nanopore structure to clear the blockage.
[0053] The nanopore can be a solid-state nanopore, namely a hole of
nanometer width, provided in a solid-state membrane. This membrane
can be the nanopore layer, or be a membrane placed upon the
nanopore layer. A solid-state nanopore can be positioned on the
nanopore layer. The nanopore can alternatively be a biological
nanopore located in a solid-state film or membrane. Further
alternatively, the nanopore layer can be formed with a well across
which a membrane, such an amphiphilic membrane or a lipid bilayer
can be formed such that a nanopore can be inserted in the membrane.
In each of these nanopore examples one nanopore can be provided for
each nanopore structure in the array.
[0054] The present inventors also sought to improve the
architecture of nanopore sensors, in particular where the
improvements could optimise the sensitivity and performance. The
inventors generally sought to achieve this by providing a structure
having nanopore structures, wherein the nanopore structures located
in the structure provide fluid communication from one side of the
structure to the other via a passage provided in each nanopore
structure. In this way the structure can separate a cis and a
trans. Each of the nanopore structures has a sensor electrode. In
order to minimise the attenuation of a signal derived from the
sensor electrode and to avoid any detriment to that signal from
noise each nanopore structure is provided with a circuit for
processing signals from the sensor electrode prior to processed
signal being communicated for further processing and/or analysis.
The circuit can be embedded in the nanopore structure. The circuit
can occupy the same footprint as the nanopore structure such that
the nanopore structure can be considered as an active pixel. A
nanopore structure having its own circuit can complement the
improved control mechanism disclosed herein by having a control
signal generated and applied locally, thus minimising the influence
of the control signal upon other nanopore structures of the
array.
[0055] Therefore, some embodiments relate to a device having
nanopore structures for sensing an analyte, the nanopore structures
arranged to separate an analyte reservoir and an outlet chamber,
each nanopore structure providing a passage for fluid connection
through the structure between the analyte reservoir and outlet
chamber, wherein each nanopore structure comprises: an electrical
transduction element; and an electronic circuit configured to
detect, and optionally amplify, a signal from the electrical
transduction element, wherein each of the structures are configured
to store, transmit, process or communicate at least a portion of
the signal to a connectable processor, or perform some combination
thereof. In some embodiments, each of the structures are configured
to at least one of store, transmit, process and communicate at
least a portion of the signal to a connectable processor.
[0056] The nanopore structures may be comprised as part of an
overall structure wherein the individual nanopore structures are
joined to each other.
[0057] The structure can be configured to separate an analyte
chamber for receiving an analyte and an outlet chamber for
collecting the analyte. Drive electrodes can be connected
respectively in the analyte reservoir and the outlet chamber for
imposing an electrical potential difference across the passages in
the nanopore structures. When provided with nanopores, the nanopore
structure can function as a nanopore sensor and the device can be a
nanopore sensing device.
[0058] Each of the nanopore structures in the array can further
comprise a compensation circuit. The compensation circuit function
can be incorporated with the other processing functions of the
circuit in the nanopore structure. The compensation circuit can
have a variable gain amplifier and/or a variable capacitor in a
feedback loop of the compensation circuit.
[0059] As described in the first aspect, the structure can have a
control terminal for applying a control signal to alter the
electrical potential difference across the nanopore structure. The
control signal can be switchably applied to the control terminal to
adjust the configurable voltage level imposed upon the pore.
[0060] The nanopore structures incorporating the circuit, which can
include a compensation circuit, can be packaged in a defined
footprint or pixel space. The array of pixel-spaced nanopore
structures can be arranged in a tessellated array.
[0061] By processing the signal from an electrical transduction
element, at least in part, within the nanopore structure itself,
the signal can be processed or managed locally. For example, the
signal can be amplified locally such that there is minimal
attenuation or noise influencing the signal before it is analysed
elsewhere. The circuit can also store the signal, signal values or
data derived from the signal. In this way, information derived from
the nanopore structure can be communicated to a processor remote
from the nanopore structure on demand. Each nanopore structure, or
circuit in the nanopore structure, can be addressable. The circuit
can be connected to an analogue to digital converter (ADC) located
off the nanopore structure.
[0062] The inventors further sought to provide a structure that, in
general, improved the manufacturability of an array of nanopore
structures, while improving sensitivity and performance. Not only
can the array of nanopore structures herein provided an improved
nanopore structure but the array of nanopore structures can
complement the integration of the control functions and local
control.
[0063] Therefore, some embodiments relate to a device having an
array of nanopore structures. The structures can be configured in a
sheet, the sheet comprising: a nanopore layer having an array of
nanopores and/or an array of wells for supporting a nanopore; and a
base layer having an array of channels, said base layer sandwiched
or laminated to the nanopore layer to form the sheet, wherein the
nanopores and/or the wells are aligned with the channels, wherein
each of the nanopore structures comprise a passage, each passage
defined at least in part by: one of the nanopores and/or one of the
wells of the nanopore layer, at one side of the passage; a channel
of the base layer at the other side of the passage; and an
electrical transduction element.
[0064] Inventive aspects can reside in the array of nanopore
structures itself. When provided with a nanopore each nanopore
structure of the array functions as a nanopore sensor. Each
nanopore structure has a through-hole defined by a nanopore, if
provided, or a well, a channel and an electrical transduction
element.
[0065] The sheet can be a substantially planar array of nanopore
structures. When the nanopore structures are provided with a
nanopore they can function as nanopore sensors. The sheet can be
configured in the device to separate a cis and a trans chamber. The
cis and the trans chamber can accommodate a fluid. The passages can
be filled with a fluid and provide a fluid connection between the
cis and the trans.
[0066] Configuring the nanopore layer and the base layer as
separate layers can improve the scalability of the sheets. The
layers can facilitate assembly of the device, thus reducing the
cost of manufacture. The layering of the sheet can bring together
the components of the nanopore structure in an efficient manner.
Moreover, by having the different components of the nanopore
structure on different layers can enable the formation or
configuration of those components to be optimised. It is often the
case that the process used in the fabrication of one component is
incompatible or detrimental to the fabrication of another
component. Furthermore, the optimal material for forming one
component can be different from the optimal material for forming
other components. By way of example, the array of nanopores and/or
an array of wells of the nanopore can be formed separately from the
base layer. The nanopore layer and base layer can comprise
different materials. The separate layers can enable the components
of the nanopore structure to be optimally configured and/or
located.
[0067] The provisions of layers can enable a layer to be
replaceable. The nanopore layer can be removably attachable. In
this way a nanopore layer can be replaced with replacement nanopore
such that the device can be recycled should, for example, the
nanopore layer become contaminated.
[0068] Each nanopore structure of the sheet is defined by the
passage. The various components of the nanopore structure i.e. the
nanopore or nanopore well, the electrical transduction element and
the channel form the passage. The nanopore layer does not have to
have a nanopore and can be provided with a nanopore. A nanopore can
be configured over the well of the nanopore layer, and in so doing
this additional nanopore over the well also forms an element of the
passage.
[0069] The electrical transduction element in each passage can be
disposed between the nanopore layer and at least a portion of the
channel. The electrical transduction element can be configured with
a connection for measuring electrical potential of the fluid at the
location of the electrical transduction element when the structure
is provided with a nanopore and a fluid is provided in the
passage.
[0070] The electrical transduction element can develop a
characteristic that is indicative of the fluidic electrical
potential at the electrical transduction element in that passage,
via fluid in the passage that connects the cis and trans. The
electrical transduction element can be an electrical connection. It
can be located in the cis or the trans reservoir, on a surface of
the nanopore structure, at a position within the passage, or other
location within the nanopore structure.
[0071] The electrical transduction element can be a device or
region of a device and/or circuit, a wire, or combination of
circuit elements, that senses the fluidic electrical potential at
the electrical transduction element of the device. Additionally, or
alternatively, the circuit can be provided as a transduction
element to develop a signal indicative of local electrical
potential.
[0072] As described, the device can have a first fluidic reservoir
and a second fluidic reservoir separated, at least in part, by the
sheet. The first fluidic reservoir can function as a cis and hold
an analyte to be analysed by the nanopore structure when provided
with a sensor. The passages of the nanopore structures of the array
connect the first fluidic reservoir to the second fluidic
reservoir. The interface between the first fluidic reservoir and
the second fluidic reservoir can be the passage or, more
specifically, the nanopore in a nanopore sensor i.e. a nanopore
structure provided with a nanopore.
[0073] The device can have drive electrodes connected in the first
and second reservoirs to impose an electrical potential difference
across the array of passages between the first and second fluidic
reservoirs.
[0074] The sheet can be substantially planar. The surfaces of the
sheet, which is the structure incorporating the array of nanopore
structures, can have a cis-surface on the nanopore layer for facing
a first fluidic reservoir and defining a cis-plane, and a
trans-surface of the base layer for facing a second fluidic
reservoir and defining a trans-plane. The array of electrical
transduction elements can be embedded, at least in part, within the
sheet between the cis-plane and the trans-plane. The electrical
transduction elements of the array can be sandwiched between the
nanopore layer and the base layer.
[0075] Each nanopore structure of the array can have a well formed
at a first end of the passage. A nanopore can be configured at the
first end of each well. The electrical transduction element can be
configured on the opposite side of the well to the nanopore. The
well can be larger in size that the nanopore and increase the
volume of fluid surrounding the nanopore. To be clear, the diameter
of the well can be greater than the diameter of the nanopore. The
nanopore can reside in a membrane that spans the well. The membrane
can be a solid-state membrane, an amphiphilic membrane or a lipid
bilayer. The nanopore defines a portion of the passage. Ingress and
egress from the well are via the nanopore and a well outlet.
[0076] The well can be configured for supporting a fluid membrane
such as a polymer membrane or lipid bilayer. The nanopore layer can
be fabricated from a different material from the base layer. By
using a different material for the nanopore layer a material can be
selected to have a surface energy that optimises the formation of a
membrane across the well for supporting a nanopore.
[0077] The electrical transduction element can be a sensor
electrode. The sensor electrode can be directly connectable to the
base or gate of a transistor device for measuring variations in
electrical potential of the fluid at the location of the electrical
transduction element when a fluid is provided in the passage. As
described herein, a nanopore structure provided with a nanopore
forming a portion of the passage functions as a nanopore sensor,
and the sensing is performed by the electrical transduction
element.
[0078] The electrical transduction elements of the nanopore
structures of the array can be connected to an edge-connector or
wire-bond. The connector can provide a connection to a measurement
circuit off-sheet i.e. separate from the array of nanopore
structures. The connector can be connected to a via that leads to a
connection at the edge of the sheet, for subsequent connection to a
measurement circuit off-sheet. The transistor device can be a field
effect transistor.
[0079] The sheet has been described thus far having a nanopore
layer and base layer. The electrical transduction element can be a
layer within the sheet or can have elements sandwiched between
layers. The sheet of the device can, however, further comprise a
sense layer having an array of the electrical transduction
elements, wherein said sense layer is sandwiched between the
nanopore layer and the base layer. The electrical transduction
element can be formed upon the sense layer. The electrical
transduction element can have an exposed portion for connection to
a fluid in the passage and an embedded portion embedded within the
sheet. Additionally, or alternatively, the electrical transduction
element can have a connection portion for connection to a
measurement circuit separate from the sheet. By incorporating the
electrical transduction element in or upon the sense layer this
enables the formation of electrical transduction element to be
separate from the manufacture of the other layers. The sense layer
can be fabricated using a different material, process and/or
techniques from the other layers.
[0080] The electrical transduction element can cover, at least in
part, a wall of the passage. The electrical transduction element
can cover, in cross-section, a portion of a wall of the channel.
The electrical transduction element can form an annulus around the
passage and/or the base of a well or cavity within the passage.
[0081] The electrical transduction elements can be formed on one
surface of the sense layer. The sense layer can be sandwiched
between the base layer and the nanopore layer with the electrical
transduction elements aligned with the nanopore or wells of the
nanopore layer and the channels of the base layer. When aligned,
the face of the sense layer can have the electrical transduction
elements exposed to the nanopore layer, such that the nanopore
layer is formed or placed upon the surface having the electrical
transduction elements; in this arrangement the electrical
transduction element can be said to face the nanopore layer.
Alternatively, when aligned, the face of the sense layer can have
the electrical transduction elements exposed to the base layer,
such that the electrical transduction element is formed or placed
upon the surface of the base layer; in this arrangement the
electrical transduction element can be said to face the base
layer.
[0082] The electrical transduction element can form, at least in
part, the surface of the sense layer around the passage and have an
exposed portion arranged to face the outlet chamber. The exposed
portion can form part of a wall of a cavity formed in the sense
layer between the well and the channel. The cavity enables a
greater area of the sensor electrode to be exposed to fluid in the
passage. This can improve the sensitivity of the sensor
electrode.
[0083] The electrical transduction element can have an aperture
forming a portion of the passage and exposed portion, wherein in
cross-section, the ratio of the size of the exposed portion of the
electrical transduction element to the size of the aperture is 1:1.
The ratio can be about 5:1.
[0084] The electrical transduction element can have an aperture
forming a portion of the passage and exposed portion, wherein in
plan-view, the ratio of the size of the exposed portion of the
electrical transduction element to the size of the aperture is 1:1.
The electrical transduction element can have an aperture forming a
portion of the passage and exposed portion, wherein the ratio is
about 5:1. The aperture can be circular.
[0085] The electrical transduction element can have a large exposed
area to increase the exposure to a fluid in the passage to increase
the sensitivity of the element to fluctuations in voltage caused by
an analyte passing over, or through, a nanopore in the passage.
[0086] The sense layer can incorporate an electronic circuit for
each nanopore structures. The circuit can be connected to the
electrical transduction element for modifying and/or processing the
signals received therefrom. By incorporating an electronic circuit
within each nanopore structure then signals from the electrical
transduction elements can be processed locally to inhibit any
attenuation of information in the signal derived therefrom and/or
inhibit any detriment to that signal from noise. Each electrical
circuit in the respective nanopore structure can process signals
from the sensor electrode prior to said processed signal being
communicated off-sheet for further processing and/or analysis. By
incorporating the circuit in the sense layer, the circuit can be
embedded in the nanopore structure. The circuit can occupy the same
footprint as the nanopore structure such that the nanopore
structure can be considered as an active pixel. A nanopore
structure having its own circuit can complement the improved
control mechanism disclosed herein by having a control signal
generated and applied locally, thus minimising the influence of the
control signal upon other nanopore structures of the array. The
circuit within the sense layer of the nanopore circuit can be a
compensation circuit.
[0087] The electronic circuit can be configured to detect changes
in voltage caused by resistance changes at a nanopore in a
respective passage when an analyte passes through the nanopore, or
adjacent said nanopore. The circuit can detect a resistance change
detected through the fluid in the sensor.
[0088] While the device has been described as suitable for sensing
an analyte it should be appreciated that the analyte is one that
can be measured using a nanopore. By way of example the analyte can
be a, protein, polymer, polynucleotide or the like.
[0089] The electronic circuit can detect resistance changes at the
nanopore when a polymer passes through the nanopore and converts it
to a voltage signal and amplifies said voltage signal. The
electronic circuit can filter the signal. The electronic circuit
can sample and/or digitise signals obtained from an electrical
transduction element.
[0090] Each nanopore structure can have a plurality of electrical
transduction elements corresponding to each respective nanopore
structure. Similarly, each nanopore structure can have a plurality
of circuits corresponding to each respective nanopore structure
and/or electrical transduction element provided in that nanopore
structure. Each of the electrical transduction elements and/or
circuits can be configured in an addressable array. Each nanopore
structure can have two or more sensor electrodes. Two or more
electrodes can be connected to a single circuit within the nanopore
structure or each sensor electrode could be connected to its own
circuit.
[0091] The array of nanopore structures can be connected to an
architecture for enabling readout from each nanopore structure
individually (which may be referred to as pixels) in a matrix
array. Each nanopore structure can have a row number and column
number.
[0092] Each electrical transduction element can have a dedicated
electronic circuit, and each electrical transduction element and
electronic circuit can be located in a footprint. The footprint can
be a pixel such that the nanopore structures are tessellated in the
array.
[0093] While each nanopore structure of the array has an electrical
transduction element and, optionally a circuit and/or a control
terminal, in light of the teaching herein it can be appreciated
that each nanopore structure can have a plurality of electrical
transduction elements and/or a plurality of circuits, each circuit
providing one or more functions. By way of example, a nanopore
structure can have an electrical transduction element for sensing,
and a corresponding circuit to process signals from that element
and have a second electrical transduction element adapted for
applying a control signal to the passage in the nanopore structure,
said second electrical transduction element having a circuit for
controllably applying said control signal.
[0094] It follows that a plurality of electrical transduction
elements can be arranged in a module having a plurality of
respective nanopore structures. The module can have a common
dedicated electronic circuit, and each of the electrical
transduction elements and electronic circuit are located in a
footprint occupied by the plurality of nanopore structures. The
module can have, for example, four nanopore structures, each having
a respective electrical transduction element, wherein each element
is connected to a common circuit. The common circuit can be
addressably connected to an external off-structure or off-sheet
electronic circuit.
[0095] The plurality of nanopore structures can be arranged in a
two-dimensional matrix. The plurality of nanopore structures can be
arranged in a tessellated pattern.
[0096] The electrical transduction element can be connected to the
base or gate of a transistor for sensing. The transistor can be a
field effect transistor.
[0097] Each of the nanopore structures can have a control terminal
for applying a control signal to alter the electrical potential
difference across the respective nanopore structure. The control
terminal can be switchably connectable to the electrical
transduction element. The control terminal can be switchably
connectable to a power supply to change the configurable voltage
level imposed upon the pore. The electrical transduction element
and connection for measuring electrical potential of the fluid can
be switchably isolatable from the control signal. The electrical
transduction element and control electrode can be physically
separate. At least a portion of the electrical transduction element
and at least a portion of the control electrode can extend in the
same plane. At least a portion of the electrical transduction
element and at least a portion of the control electrode form, at
least in part, the base of a well. At least a portion of the
electrical transduction element and at least a portion of control
electrode can extend perpendicularly from one another. At least a
portion of the control electrode can be configured, at least in
part, in the channel. The surface area of the electrical
transduction element exposed to the passage can be less than the
surface area of the control electrode exposed to the passage.
[0098] The device herein can be configured with a conductive guard
configured in at least one of the nanopore layer, base layer or
sensing layer. The conductive guard can extend between at least one
of the electrical transduction element, and signal conductors
connected thereto, and parasitic conductive elements in the
nanopore layer, base layer or sense layer to inhibit parasitic
capacitance from influencing the measurements obtained from the
connection. A buffered version of the input signal can be applied
to the guard conductor. As a result, there is no voltage difference
across the capacitance from the input signal conductor to the
conductive substrate.
[0099] The conductive guard can include, at least in part, an
insulated guard conductor having and an insulating layer. The
conductive guard can be configured to extend, at least in part,
between the base layer and the channel.
[0100] The inventors have further considered the operation and
manufacturability of the devices disclosed herein.
[0101] Some embodiments relate to a method of operating a device as
described for nanopore sensing, the method comprising:
translocating analyte through an array of nanopores under a
potential difference applied across the array, measuring a change
in the fluidic electrical potential at each nanopore by means of
respective electrical transduction elements of and responsive to
the measurement, applying a control signal to a control terminal of
an electrical transduction element to alter the electrical
potential difference across the nanopore. Therefore, some
embodiments relate to a method of operating a device for nanopore
sensing, the method comprising: imposing an electrical potential
difference across an array of nanopore sensors disposed in a
structure separating an analyte reservoir and an outlet chamber,
each nanopore sensor having a passage for providing a fluid
connection between the analyte reservoir and the outlet chamber;
providing an analyte for analysis by the nanopore sensors, each
nanopore sensor having an electrical transduction element for
measuring a change in the fluidic electrical potential at the
electrical transduction element of that nanopore sensor when an
analyte is induced through a nanopore of the nanopore sensor; and
applying a control signal to a control terminal of an electrical
transduction element of a nanopore sensor of the array to alter the
electrical potential difference across that nanopore sensor.
Fluidic electrical potential can be measured at the electrical
transduction element. The fluidic electrical distribution across
that nanopore structure can be altered when the device is provided
with a fluid. In operation, a fluid resides in the reservoir,
chamber and passages of the nanopore structure. The fluid in the
reservoir, chamber and passages of the nanopore structure can be
different fluids.
[0102] The electrical potential difference imposed across the array
serves to induce an analyte through, or at least in to, the
passage. An analyte to be analysed is provided in the analyte
reservoir and induced to the outlet chamber, which is achieved by
the drive electrodes. The situation can, however, be reversed in
that an analyte can be provided in the outlet chamber or an analyte
in the outlet can be induced by the drive electrode in to the
analyte reservoir e.g. by changing the potential difference between
the drive electrodes.
[0103] In each case, the electrical transduction elements of each
nanopore structure, which are provided with nanopores to function
as nanopore sensors, can measure a change in the fluidic electrical
potential. The array of nanopore structures is dimensioned such
that the electrical transduction element of one nanopore sensors is
inhibited from detecting an analyte passing through a nanopore in a
neighbouring nanopore structure.
[0104] A control signal can be applied to an element to alter the
electrical potential difference across the nanopore sensor in which
said element resides.
[0105] The control terminal connected to the electrical
transduction element can be switchably connected to the control
terminal of the electrical transduction element for applying the
control signal thereto. Additionally, or alternatively, the device
can be operated to isolate any sensing circuitry from the
electrical transduction element to inhibit damage to said circuitry
while the control signal is applied.
[0106] The method can include analysing characteristics of the
change in the electrical potential locally at a nanopore sensor and
applying the control signal to that nanopore sensor in response to
predetermined characteristics. The method can apply a control
signal to an electrical transduction element of a nanopore sensor
to alter the potential difference imposed by the drive electrodes
at that nanopore sensor. The change in potential difference can
induce movement of an analyte or a free-moving nanopore, which can
be charged.
[0107] The control signal can perform a plurality of operations
including, but not limited to: inducing pore insertion in to a
membrane formed across the passage; unblocking a nanopore;
rejecting an analyte; altering the rate of translocation of an
analyte through that nanopore. In forming a device having nanopore
structures for sensing an analyte, the method of forming comprises:
forming nanopore structures in a structure and arranging said
structure to separate an analyte reservoir and an outlet chamber of
the device such that each nanopore structure provides a passage for
fluid connection through the structure between the analyte
reservoir and outlet chamber; and fabricating in each nanopore
structure: an electrical transduction element; and an electronic
circuit configured to measure a signal from the electrical
transduction element, wherein each of the nanopore structures are
configured to at least one of store, transmit, process and
communicate at least a portion of the measured signal, or
information derived therefrom, to a connectable processor.
[0108] Fabricating an electronic circuit in each nanopore structure
can enable measurements to be made at the electrical transduction
element at that nanopore structure when provided with a nanopore to
function as a sensor.
[0109] While measurements taken from a sensor can be communicated
directly to an off-structure circuit for analysis the ability to
locally process or condition the signal or information therefrom
can improve noise performance, data management or amplification. By
way of example, a circuit located within the nanopore structure can
amplify a signal received from the electrical transduction element
and, by amplifying the signal locally the level of noise amplified
is minimised. If, for example, a signal received from the
electrical transduction element were to be communicated
off-structure before amplification for analysis the exposure of
said signal to noise would be increased and subsequently amplified
thus reducing the signal to noise ratio.
[0110] The method can further include configuring an analyte
reservoir for receiving an analyte and an outlet chamber for
collecting the analyte and configuring the nanopore layer to
separate the analyte reservoir and outlet chamber. The structure
can separate the cis and the trans of the device.
[0111] The method can further include configuring drive electrodes
connected respectively in the analyte reservoir and the outlet
chamber for imposing an electrical potential difference across the
passage of the nanopore structures. The imposed electrical
potential difference can be common across the plurality of nanopore
structures. Multiple drive electrodes can be provided to achieve a
common potential difference across the array of nanopore
structures.
[0112] The method can further comprise configuring the electronic
circuits with a switchable connection for applying a signal to a
respective control terminal of the electrical transduction element
for altering the electrical potential imposed by the drive
electrodes across each respective nanopore structure.
[0113] The method can further comprise forming a control electrode
in the passage of each nanopore sensor, said control electrode
selectably connectable to a signal for altering an electrical
potential imposed by the drive electrodes across each respective
nanopore structure.
[0114] In fabricating a device having nanopore structures for
sensing an analyte, the method of fabrication comprises forming a
device having an array of nanopore structures configured in a
sheet, including arranging the sheet to separate an analyte
reservoir and an outlet chamber of the device such that each
nanopore structure provides a passage for fluid connection through
the structure between the analyte reservoir and outlet chamber, the
method comprising: forming a nanopore layer having an array of
nanopores and/or an array of support structures, such as wells, for
supporting a nanopore; forming an array of electrical transduction
elements; forming a base layer having an array of channels, said
base layer sandwiched or laminated to the nanopore layer to form
the sheet such that the nanopores and/or the wells are aligned with
the electrical transduction elements and channels; and providing a
passage through each of the nanopore structures such that each
passage is defined at least in part by: one of the nanopores and/or
one of the wells of the nanopore layer, at one side of the passage;
a channel of the base layer at the other side of the passage; and
an electrical transduction element.
[0115] Aligning the nanopore layer, base layer and array of
electrical transduction elements can include sandwiching the array
of electrical transduction elements between the nanopore layer and
the base layer. The step of sandwiching can include bonding or
otherwise connecting the two layers.
[0116] The method can further comprise forming cavities adjacent at
least a portion of each of the electrical transduction elements.
These cavities can increase the area of the element exposed to a
fluid in the passage.
[0117] The method can further comprise: forming the array of
electrical transduction elements on a sense layer; and sandwiching
the sense layer between the nanopore layer and the base layer.
[0118] The method can further comprise: forming the array of
electrical transduction elements on a sense layer; fabricating an
array of electronic circuits in the sense layer, said circuits
connected to respective electrical transduction elements for
modifying and/or processing the signals received therefrom; and
sandwiching the sense layer between the nanopore layer and the base
layer.
[0119] The method can further comprise arranging the electrical
transduction element to have: (i) an exposed portion for connection
to a fluid in the passage, and (ii) an embedded portion embedded
within the structure, and/or (iii) a connection portion for
connection to a measurement circuit separate from the
structure.
[0120] The method can further comprise forming a conductive guard
in at least one of the nanopore layer, base layer or sense layer,
said conductive guard configured to extend between at least one of
the electrical transduction elements and signal conductors
connected thereto and parasitic conductive elements in at least one
of the nanopore layer, base layer or sense layer to inhibit
parasitic capacitance from influencing the measurements obtained
from the connection.
[0121] The method can further comprise providing for each nanopore
structure a buffer, said buffer connecting the output of the
electrical transduction element of that nanopore structure to a
conductive guard.
[0122] The method can further comprise providing amphiphilic
membranes in each of the nanopore structures of the array and
inserting a biological nanopore in said membranes.
[0123] The method can include removably attaching the structure
and/or removing the nanopore layer and replacing it with another
nanopore layer. In this manner the device can be recycled.
[0124] Some embodiments relate to a device having a plurality of
nanopore structures configured in a sheet, the sheet comprising: a
nanopore layer having a plurality of nanopores and/or a plurality
of wells for supporting a plurality nanopores; and a base layer
having a plurality of channels, said base layer laminated to the
nanopore layer to form the sheet, wherein the plurality of
nanopores and/or the plurality of wells are aligned with the
plurality of channels, wherein two or more of the nanopore
structures each comprise a passage defined at least in part by: one
of the nanopores and/or one of the wells of the nanopore layer, at
one side of the passage; a channel of the base layer at the other
side of the passage; and an electrical transduction element.
[0125] Some embodiments relate to a method of operating a device
for nanopore sensing, the method comprising: imposing an electrical
potential difference across a plurality of nanopore sensors
disposed in a structure separating an analyte reservoir and an
outlet chamber, two or more of the nanopore sensors each having a
passage for providing a fluid connection between the analyte
reservoir and the outlet chamber; providing an analyte for analysis
by the nanopore sensors, the two or more nanopore sensors each
having an electrical transduction element for measuring a change in
the electrical potential at the electrical transduction element of
that nanopore sensor when an analyte is induced through a nanopore
of that nanopore sensor; and applying a control signal to a control
terminal of an electrical transduction element of one of the two or
more nanopore sensors to alter the electrical potential difference
across that nanopore sensor.
[0126] Some embodiments relate to a method of forming a device
having nanopore structures for sensing an analyte, the method
comprising: forming the nanopore structures in a structure and
arranging said structure to separate an analyte reservoir and an
outlet chamber of the device such that two or more nanopore
structures each provide a passage for fluid connection through the
structure between the analyte reservoir and outlet chamber; and
fabricating in each of the two or more nanopore structures: an
electrical transduction element; and an electronic circuit
configured to receive a signal from the electrical transduction
element, herein the electronic circuit is configured to at least
amplify and/or store the signal, or information derived
therefrom.
[0127] Some embodiments relate to a method of forming a device
having an array of nanopore structures arranged in a sheet that is
configured to separate an analyte reservoir and an outlet chamber
of the device such that two or more nanopore structures each
provide a passage for fluid connection through the sheet between
the analyte reservoir and outlet chamber, the method comprising:
forming a nanopore layer having an array of nanopores and/or an
array of wells for supporting a nanopore; forming an array of
electrical transduction elements; forming a base layer having an
array of channels, said base layer laminated to the nanopore layer
to form the sheet such that the array of nanopores and/or the array
of wells are aligned with the array of electrical transduction
elements and array of channels, wherein each passage of the two or
more nanopore structures is defined at least in part by: one of the
nanopores and/or one of the wells of the nanopore layer, at one
side of the passage; a channel of the base layer at the other side
of the passage; and an electrical transduction element.
[0128] Many aspects have been described herein, and elements of
different aspects can, in light of the teaching herein, be
combined. Many further aspects are, therefore, implicit in light of
the teaching of the description and the figures, which often
combine two or more of the aspects described herein. In general,
the different aspects may be combined together in any
combination.
[0129] Other advantages and novel features of the present invention
will become apparent from the following detailed description of
various non-limiting embodiments of the invention when considered
in conjunction with the accompanying figures. In cases where the
present specification and a document incorporated by reference
include conflicting and/or inconsistent disclosure, the present
specification shall control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0130] Non-limiting embodiments of the present invention will be
described by way of example with reference to the accompanying
figures, which are schematic and are not intended to be drawn to
scale. In the figures, each identical or nearly identical component
illustrated is typically represented by a single numeral. For
purposes of clarity, not every component is labeled in every
figure, nor is every component of each embodiment of the invention
shown where illustration is not necessary to allow those of
ordinary skill in the art to understand the invention. In the
figures:
[0131] FIG. 1 depicts a cross sectional view of an example nanopore
sensor of the related art;
[0132] FIG. 2 is a cross-section of a single sensor electrode and
corresponding biological nanopore within a nanopore array
configured in a structure, and connected via a wire to a
measurement circuit, according to some embodiments;
[0133] FIG. 3(a) is an alternative cross-section of a single sensor
electrode, and corresponding biological nanopore, the sensor
electrode configured on a sensor layer that is sandwiched between a
nanopore layer and a base layer within a portion of a structure of
a nanopore sensor array, wherein the sensor electrode is connected
via a wire to an electronic circuit, according to some
embodiments;
[0134] FIG. 3(b) is comparable to FIG. 3(a) wherein the sensor
layer incorporates electronic circuitry;
[0135] FIG. 4(a) is a schematic view of the layout of a nanopore
sensor of FIG. 3(b) indicating the position of the well with
respect to the electronic circuitry, according to some
embodiments;
[0136] FIG. 4(b) shows two adjacent sensor electrodes before a pore
is added;
[0137] FIG. 4(c) and FIG. 4(d) are examples of portions of a
structure having an array of the nanopore sensors of FIG. 4(a);
[0138] FIG. 4(e) is a schematic showing how a structure can be
arranged to separate two chambers in a device, according to some
embodiments;
[0139] FIG. 4(f) is an alternative layout of four nanopore
structures;
[0140] FIG. 5(a) illustrates example circuitry for interfacing with
an electrical transduction element, according to some
embodiments;
[0141] FIG. 5(b) illustrates example circuitry for interfacing with
an electrical transduction element, according to some
embodiments;
[0142] FIG. 5(c) illustrates example circuitry for interfacing with
an electrical transduction element, according to some
embodiments;
[0143] FIG. 5(d) illustrates example circuitry for interfacing with
a plurality of electrical transduction elements, according to some
embodiments;
[0144] FIG. 6(a) depicts an electrical transduction element in plan
view, according to some embodiments;
[0145] FIG. 6(b) depicts a cross section of a sensor electrode and
control electrode configured in a sensor of the array, according to
some embodiments;
[0146] FIG. 6(c) depicts a cross section of a sensor electrode and
control electrode configured in a sensor of the array, according to
some embodiments;
[0147] FIG. 6(d) depicts a cross section of a sensor electrode and
control electrode configured in a sensor of the array, according to
some embodiments;
[0148] FIG. 6(e) depicts a cross section of a sensor electrode and
control electrode configured in a sensor of the array, according to
some embodiments;
[0149] FIG. 7(a) depicts, respectively, two schematic circuits
illustrating the parasitic capacitance in the array with and
without guarding;
[0150] FIG. 7(b) depicts an alternative cross-sectional view to
that shown in FIG. 2, in which a guard conductor is configured in
the structure and connected via an additional wire to the
measurement circuit, according to some embodiments;
[0151] FIG. 7(c) depicts an alternative cross section of a nanopore
structure in which guarding is implemented; and
[0152] FIG. 7(d) depicts an alternative cross section of a nanopore
structure in which guarding is configured.
DETAILED DESCRIPTION
[0153] In overview, devices for improved nanopore sensing are
described. An example device can have a structure arranged to
separate an analyte reservoir and an outlet chamber. The structure
can have an array of nanopore structures, each nanopore structure
comprising a passage for fluid connection through the structure
between the analyte reservoir and outlet chamber. Control terminals
can be included wherein each terminal connects to a respective
nanopore structure for applying a control signal to alter the
electrical potential difference across that nanopore structure. In
further aspects, an improved nanopore structure for sensing an
analyte can include an electronic circuit configured to detect a
signal from an electrical transduction element, and wherein each of
the structures may be configured to at least one of store,
transmit, process and communicate at least a portion of the signal
to a processor.
[0154] Some embodiments of a device for improved nanopore sensing
have an array of nanopore structures configured in a sheet, the
sheet comprising: a nanopore layer having an array of nanopores
and/or an array of wells for supporting a nanopore; and a base
layer having an array of channels, said base layer sandwiched to
the nanopore layer to form the sheet, wherein the nanopores and/or
the wells are aligned with the channels, wherein each of the
nanopore structures comprise a passage, each passage defined at
least in part by: one of the nanopores and/or one of the wells of
the nanopore layer, at one side of the passage; a channel of the
base layer at the other side of the passage; and an electrical
transduction element.
[0155] Inventive aspects further relate to a method of operating a
device for nanopore sensing, the method including: applying a
control signal to a control terminal of an electrical transduction
element of a nanopore sensor of the array to alter the electrical
potential difference across that nanopore sensor.
[0156] Additional embodiments relate to methods of forming a device
having nanopore structures for sensing an analyte. An example
method may include fabricating in each nanopore structure: an
electrical transduction element; and an electronic circuit
configured to measure a signal from the electrical transduction
element.
[0157] In further detail, FIGS. 2 to 4(a) are sectional views of a
portion of a structure 100 having a nanopore structure 104
incorporated therein. In some implementations, the structure 100
has an array of nanopore structures 104, each nanopore structure
adapted to support a nanopore 116. The nanopore structures of the
device can function as nanopore sensors when configured with a
nanopore. A nanopore sensor herein includes a nanopore structure
having a nanopore. In some embodiments, a nanopore sensor herein is
a nanopore structure having a nanopore.
[0158] FIGS. 4(b) to 4(f) illustrate that a plurality of the
nanopore sensors 102 shown in FIGS. 2 to 4(a) can be arranged as
part of an array of nanopore structures 104.
[0159] The structure 100, which may take the form of a sheet,
incorporates the array of nanopore sensors (n.b. only one nanopore
sensor of the array is shown) and can be configured within a device
or device for analysing an analyte, as shown in FIG. 4(e).
[0160] The structure 100 separates the analyte reservoir 106 for
receiving an analyte and an outlet chamber 108. The structure 100
has a nanopore layer 110 configured upon a base layer 112, which
together forms at least a portion of the structure having a
plurality of nanopore sensors 102. Each nanopore sensor 102 in the
array of nanopore structures 104 has a passage 114 configured to
extend through the nanopore layer and base layer of the array for
connecting the analyte chamber and outlet chamber.
[0161] The nanopore layer 110 of each nanopore sensor 102 may
optionally be provided with a nanopore 116 in a membrane 118
supported by the nanopore layer. The nanopore layer 110 of each
nanopore sensor 102 may optionally be provided with a nanopore 116
in a membrane 118 supported between the pillars of the nanopore
layer. Alternatively, the nanopore can be a so-called solid state
nanopore, namely a nanometer sized through-hole provided in a
solid-state support layer. Further alternatively, the nanopore can
be a so-called hybrid nanopore, namely a biological nanopore
provided in an aperture of a solid-state membrane. Either way, the
nanopore is provided in a membrane proximal the first end 120, or
pore end, of the passage 114 (e.g. at the top of the sensor as
shown).
[0162] The base layer 112 has a channel 122 proximal an opposite
end 124, or channel end, of the passage 114 to the first end 120
(e.g. at the bottom of the sensor as shown). The passage 114
extends through the nanopore structure connecting one side to the
other. The channel 122 forms part of the passage. The channel is
structurally and geometrically configured to function as a fluidic
resistor. This can be achieved by defining the aspect ratio of the
channel. Additionally, or alternatively other techniques for
implementing fluidic resistance in the channel can be used. The
channel can be configured such that the resistance of the channel
and the nanopore are substantially matched, when the passage is
occupied by fluid, and relatively high relative to the resistance
of fluid in the cis and trans reservoirs such that the resistance
of the reservoirs does not appreciably influence the measurements.
In other words, the channel is configured as a fluidic resistor to
approximate the resistance of the nanopore means that the
resistance of other circuit elements such as the of the fluid in
the reservoirs has less significance and does not require
compensation to account for it when measurements are taken.
[0163] The fluidic resistance of the channel 122 can be varied by
varying its dimensions, in particular its aspect ratio and by
varying the ionic concentrations of the fluids in the analyte
reservoir 106 and the outlet chamber 108. For example, the channel
122 can be configured with a high aspect ratio to increase the
resistance. Additionally, or alternatively, the fluid in the
channel can have a lower ionic concentration compared to the fluid
in the cis and trans to increase the channel's resistance.
Maintaining a higher ionic concentration higher in the cis and the
trans improves the signal to noise ratio.
[0164] In some embodiments, the aspect ratio can, for example, be
between about 100:1 to about 2000:1, which is a ratio of channel
length to channel diameter or largest transverse dimension. In some
embodiments, the ionic concentration difference may be between
about 1:1 to about 2000:1, for example around 1000:1, which is a
ratio of ionic concentration in the cis and/or trans reservoirs to
the ionic concentration in the channel.
[0165] The signal-to-noise ratio may be optimised by selecting the
fluidic resistance of the channel 122 to be equal to the resistance
of the nanopore 116. However, this is not essential and the fluidic
resistance of the channel 122 may be varied from this value to take
account of other factors, while still obtaining an acceptable
signal-to-noise ratio. An acceptable signal-to-noise ratio may be
achieved with the fluidic resistance of the channel 122 being
significantly less than the resistance of the nanopore 116, for
example with the fluidic resistance of the channel 122 being 10% or
less of the resistance of the nanopore 116, for example 2% of the
resistance of the nanopore 116 or less. In some embodiments, a
lower limit on the fluidic resistance of the channel 122 may be set
by the desired signal to noise ratio. In other embodiments, a lower
limit on the fluidic resistance of the channel 122 may be set by
the threshold for crosstalk between adjacent channels during
flicking (as described below). That is, the fluidic resistance of
the channel 122 is desirably significantly greater than the
resistance from the end of the channel 122 to the electrical
transduction element to prevent these resistances forming a voltage
divider which applies a fraction of the applied voltage to adjacent
channels 122.
[0166] Other factors that may be considered in the selection of the
fluidic resistance of the channel 122 are as follows.
[0167] As the fluidic resistance of the channel 122 increases, the
diffusion of ions decreases, causing increased depletion of ions
near the pore, and thereby causing a decay of the signal over the
timescale of a typical event over which a signal is obtained. In
order to increase the limit on read length caused by this effect,
the fluidic resistance of the channel 122 may be reduced. In many
embodiments, this factor may place an upper limit on the fluidic
resistance of the channel 122.
[0168] As the fluid channel 122 and the nanopore 116 act as a
voltage divider, the voltage across the nanopore 116 is affected by
the current flowing through it. As the fluidic resistance of the
channel 122 increases, the variation in the voltage across the
nanopore 116 increases, which can complicate signal processing. In
order to limit this effect, the fluidic resistance of the channel
122 may be reduced.
[0169] Channels having lower fluidic resistances are easier to
fabricate, and may open up alternative fabrication techniques that
improve yield or reduce cost.
[0170] Reducing the fluidic resistance of the channel 122 may
increase bandwidth or provide leeway for additional capacitance in
the passage.
[0171] Taking into account these factors, the fluidic resistance of
the channel 122 may be less than the resistance of the nanopore
116, typically at most 50%, or at most 25% of the resistance of the
nanopore 116. In some embodiments, the optimal fluidic resistance
of the channel 122 may be around 10% of the resistance of the
nanopore 116.
[0172] When reducing the ratio of the fluidic resistance of the
channel 122 to the resistance of the nanopore 116, the signal to
noise ratio does not scale directly with that resistance ratio. For
example, in some embodiments when the fluidic resistance of the
channel 122 is around 10% of the resistance of the nanopore 116,
then the signal to noise ratio is around 30% of its optimal
value.
[0173] The channel can be formed in a wafer, and after a passage is
formed therethrough an oxide layer can be used to reduce the
diameter of the passage through the base layer, thus enabling the
amount of oxidisation to adjust the aspect ratio.
[0174] A sensor electrode 126 is disposed between the nanopore 116
and at least a portion of the channel 122. The sensor electrode 126
forms an electrical transduction element in this example. More
generally, the sensor electrode 126 could be adapted to be formed
as an electrical transduction element of any of the various types
disclosed in WO2016/127007. The sensor electrode 126 is exposed, at
least in part, to the passage 114 in the nanopore sensor 102, and
configured with a connection 128 for measuring electrical potential
of the fluid at the location of the sensor electrode 126 when a
fluid is provided in the passage. The connection 128 may attach to
a control terminal 129. Together with the nanopore layer and base
layer, the sensor electrode defines the walls of the passage 114.
The connection 128 can be a wire-bond to a separate electronic
circuit 130, said circuit configured to apply control signals
(e.g., bias voltages) and/or analyse signals obtained from the
sensor electrode 126.
[0175] The analyte chamber or cis 106 can function as a first
fluidic reservoir, while the outlet chamber or trans 108 can
function as a second fluidic reservoir. The structure 100 can
separate, at least in part, the cis and the trans and the passage
114 of a sensor 102 connects the first fluidic reservoir to the
second fluidic reservoir.
[0176] In use, the passage 114 of each nanopore sensor 102 is
occupied by a fluid. Further, drive electrodes 132 in the cis and
trans comprise at least one respective cis electrode 132a and at
least one respective trans electrode 132b configured to impose an
electrical potential difference across the fluidic passages 114 of
the nanopore structures in the array of nanopore structures 104
between the first and second fluidic reservoirs.
[0177] The structure 100 can be substantially planar. The array of
nanopore structures 104 can be substantially planar. Non-planar
configurations are envisaged by the inventors but not described
herein. The sensors 102 in the array have a cis-surface 134 of the
nanopore layer 110 arranged facing the first fluidic reservoir 106
and defining a cis-plane 136, and a trans-surface 138 of the base
layer 112 for facing a second fluidic reservoir 108 and defining a
trans-plane 140. This cis-plane 136 and trans-plane 140 are
indicated by the hashed line in FIGS. 2, 3(b) and 7(b). The sensor
electrode 126 is embedded within the structure between the
cis-plane and the trans-plane. The nanopore 116 can lie
substantially on the cis-plane 136 at the first end 120 of the
passage while the opposite end 124 of the passage can lie
substantially on the trans-plane 140.
[0178] As shown in the assembly of FIG. 2, the sensor electrode 126
can, at least in part, be embedded between in the structure 100
between the nanopore layer 110 and the base layer 112. In other
words, the sensor electrode 126 is sandwiched or laminated between
the nanopore layer and the base layer.
[0179] The nanopore layer 110 has a well 142 formed at the first
end of the passage. In the example of FIG. 2 the nanopore 116 is
configured at the first end of the passage 114, on one side of the
well, substantially on the cis-plane 136. The sensor electrode 126
can be configured (e.g., is configured) on the opposite side of the
well to the nanopore, as shown. The well 142 is shown as a
cup-shaped recess with a membrane, shown in cross-section, across
its rim. The well is configured to receive an analyte that has
passed through a nanopore. Note that the well 142 is fluidly
connected to the channel 122 via a well aperture 142a, which can be
described as a well outlet. The aperture 142a functions to enable
the analyte chamber to be fluidly connected to the outlet chamber.
The aperture 142a does not function as a nanopore. In some
implementations, the aperture 142a is configured to enable an
analyte to pass therethrough uninhibited i.e. without influencing
movement of the analyte from the cis to the trans.
[0180] Although the aperture provides a fluid connection between
the cis 106 and trans 108 an analyte that has passed through the
nanopore 116 can remain in the well 142. The well 142 and channel
122 can be considered part of the outlet chamber 108. In the
example shown in FIG. 2, the aperture is centrally located at the
base of the well within the sensor electrode 126.
[0181] The well 142, and more generally the nanopore layer 110, is
configured for supporting a fluid membrane 118 such as a polymer
membrane or lipid bilayer. The nanopore layer 110 can be fabricated
separately from the base layer 112. The nanopore layer can be
formed from a different material from the base layer, and may be
formed from a material other than a polymer or lipid bilayer. The
nanopore layer can be formed from at least one of: a
photolithographically prepared material; a moulded polymer; or a
laser etched plastic.
[0182] According to some embodiments, the sensor electrode 126 is
directly connectable to the base or gate of a transistor device for
measuring electrical potential of the fluid at the location of the
sensor electrode 126 when a fluid is provided in the passage. In
some cases, the sensor electrode 126 can be connected to an
edge-connector or wire-bond, optionally by a conductive via and/or
interconnect, to a measurement circuit 130 off-structure. The
transistor device can be a field effect transistor and the
configuration of the transistor and its optional integration in to
the structure is described in an example below. In some
embodiments, the transistor device (not shown) can be located in
the electronic circuit 130.
[0183] The nanopore sensor 102 shown in FIG. 2 is an example in
which the sensor electrode can be formed upon the base layer 112.
While the sensor electrode 126 of FIG. 2 can be formed on the base
layer 126 directly it can, alternatively, be formed separately upon
a sense layer 144, as depicted in FIG. 3(a) and FIG. 3(b). After
forming the sensor electrode 126 on a sense layer, the sense layer
may then be sandwiched (e.g., is sandwiched) between the nanopore
layer 110 and base layer 112, resulting in the structure shown in
FIG. 3(a) in some implementations.
[0184] The sense layer 144 can be fabricated in a similar manner to
the base layer 112, wherein a wafer has passages formed
therethrough, substantially perpendicular to the surfaces of the
wafer.
[0185] Alternatively, the wafer can be post-processed to open up
the passage. The passages and/or channels 122 can be formed using
techniques such as photolithography or deep reactive-ion etching
(DRIE) or combinations thereof. The wafer can be enclosed by an
oxide layer. The wafer can be a CMOS wafer. The sensor electrode
126 can be formed on the sense layer around the passages on one
side of the sense layer, according to some embodiments. The
passages through the sense layer 144, and the sensor electrode 126
formed around these passages, are arranged to have a pitch or
layout that results in alignment with channels 122 on the base
layer 112. When secured together, the passages of the sense layer
144 are aligned with the channels 122 of the base layer. In some
cases, the nanopore layer 110 may be a polymer that is moulded or
lithographically etched. The base layer 112 may comprise a
semiconductor material, such as silicon. The sense layer 144 may
comprise semiconductor materials and may be part of a CMOS
wafer.
[0186] By way of example, the nanopore layer 110 may be made of
polymer, which can be moulded or lithographically etched; the base
layer 112 may be formed of a silicon wafer; and/or the sensor layer
144 may be a CMOS wafer.
[0187] The sense layer 144 can be aligned and bonded to the base
layer 112 in one of two orientations. In one orientation (not
shown) the sensor electrode remains fully exposed after
bonding--that is to say that the sensor electrode: is not
sandwiched between the sense layer; is distal from the base layer
after the sense layer is secured to the base layer; and is
subsequently sandwiched between the sense layer and the nanopore
layer. In the other orientation, as shown in FIG. 3(a), the sensor
electrode 126 is formed on top of a sense layer which is then
inverted before bonding to the base layer such that the sensor
electrode faces down, as viewed, and is sandwiched between the
sense layer 144 and the base layer 112. Prior to bonding in this
configuration, a section of the oxide layer on the base layer
around the channel can be etched away or otherwise removed to
create a cavity 146 such that there is an increased area of the
sensor electrode exposed to the passage 114 after bonding. The area
of exposed electrode can be maximised to increase contact with a
fluid in the passage.
[0188] The wells 142 of the nanopore layer 110 are aligned with the
passages and sensor electrodes 126 are bonded to the sense layer
with the sensor electrodes 126 sandwiched therebetween. Looking at
FIG. 3(b), and noting that the sensing layer 144 is fabricated from
the bottom upwards, the last stage is the application of the sensor
electrode 126 on top. When assembled, the sensor layer 144 is
flipped over such that the sensor electrode 126 that was on top is
now facing downwards, as shown in FIG. 3(b). The space 146 etched
out of the base layer 112 oxide layer (the grey part) means that
the sensor electrode is sufficiently exposed.
[0189] In some embodiments, the sensor electrode 126 remains
exposed, at least in part, to the passage and configured with a
connection for measuring electrical potential of the fluid at the
location of the sensor electrode near the nanopore or at the
nanopore when a fluid is provided in the passage. Arrangements of
the sensor electrode 126--which minimise its surface area openly
facing one of the analyte or outlet chambers (e.g. the arrangements
of FIG. 2 or FIG. 3(a))--function to limit exposure to the analyte
chamber 106 or outlet chamber 108 to inhibit contamination of the
surface of the sensor electrode 126. One such example is shown in
FIG. 3(a) that shows the sensor electrode substantially partially
enclosed in the passage. Before population with a fluid, or during
the formation of an amphiphilic membrane for supporting a
biological nanopore, the surface of the sense electrode 126 can be
exposed to fluids that could contaminate the surface of the
electrode, thus if there is a contamination risk then it can be
mitigated.
[0190] In one configuration, at least a portion of the sensor
electrode 126 can be arranged to face away from the well 142 toward
the channel 120, as shown in FIG. 3(a). An exposed portion of the
sensor electrode provides a connection to a fluid in the passage
114 for sensing fluctuations in the fluidic electrical potential at
the sensor electrode when an analyte passes through the pore. The
sensor electrode 126 also can have (e.g., has) an embedded portion
embedded within the structure. The sensor electrode 126 can also
have a connection portion 128, such as a wire-bond, for connection
to an electronic circuit 130, such as a measurement circuit or
control circuit, which can be separate from the structure as shown
in FIG. 3(a).
[0191] In each of the examples, the sensor electrode 126 can be
configured in various configurations for exposure to a fluid within
the passage and can, at least one of: cover, at least in part, a
wall of the passage; cover, in cross-section, a portion of a wall
of the passage; form an annulus around the passage; form, at least
in part, the surface of the base layer or the sense layer around
the passage and have an exposed portion arranged to face the
analyte chamber; form, at least in part, the surface of the sense
layer around the passage and have an exposed portion arranged to
face the outlet chamber. In particular, a cavity 146 can be formed
in the passage to create a region that enables the area of sensor
electrode exposure to be increased and come in to contact with an
increased amount of fluid. The cavity 146 is formed by recesses
formed in the base layer 112 and/or sense layer 144 before the base
and sense layer are aligned and connected. While the sensor
electrode 126 can have a minimal degree of exposure to the fluid in
a well, such as in the form of a nanowire, the inventors have
proposed the examples herein to optimise performance of the
nanopore sensor 102 and improve manufacturability.
[0192] As shown in FIGS. 2 and 3(a) the sensor electrode 126 is
substantially planar and shaped to accommodate the passage 114. In
other words, the sensor electrode 126 is configured to enable
uninhibited fluid communication between the cis 106 and trans 108,
which can be achieved by either (i) shaping the sensor electrode to
extend around the passage 114 or well aperture 142a, and/or (ii)
forming a sensor aperture 148 in the sensor electrode.
[0193] The footprint of the exposed portion of the sensor electrode
126 can be any shape. The well 142 of FIG. 2 and the cavity 146 of
FIG. 3(a) can be cylindrical such that the floor of the well is
circular, or a planar surface of the cavity is curved. These
configurations result in the exposed portion of the sensor
electrode being circular or disc-shaped. In the examples shown a
sensor aperture 142a, 148 is provided such that the footprint of
the exposed portion is shaped like an annulus. The exposed area of
the sensor electrode can be maximised, which can mean covering at
least one face or surface of the well 142 and/or cavity 146.
[0194] The sensor electrode 126 and the sensor aperture 148 are
shown as circular but could have other shapes. In some embodiments
having circular shapes, the ratio of the radius of the exposed
portion of the sensor electrode 126 to the radius of the sensor
aperture 148 can be in a range from about 2:1 to about 100:1 (e.g.,
the ratio can be about 2:1) or in a range from about 10:1 to about
100:1. In the case of non-circular shapes, the ratio of the square
roots of the areas may have the same values.
[0195] Alternatively, the area of exposed portion of the sensor
electrode 126 can be expressed in relation to the ratio between the
area or footprint as viewed of the sensor aperture 148 can be about
1:1, or about 10:1 or about 100:1
[0196] By way of example, the sensor electrode 126 may have a
diameter (or maximum dimension) in a range from 10 .mu.m to 50
.mu.m and the sensor aperture 148 may have a diameter (or maximum
dimension) in a range upwards from 0.5 .mu.m. The sensor aperture
148 does not function as a sensor so its size does not have an
upper limit within the bound that it is desirable to minimise the
restriction of the remaining area of the sensor electrode 126.
[0197] The sensor electrode 126 may be formed from a suitable
conductive material. In some cases, the sensor electrode 126 may be
formed using platinum. In some implementations, the sensor
electrode 126 may be formed using gold.
[0198] While FIGS. 2 and 3(a) have a sensor electrode 126 having a
connection 128 to a separate electronic circuit 130, FIG. 3(b)
illustrates that the structure 100 and array of nanopore structures
104 can accommodate an integrated circuit 150. The integrated
circuit can incorporate one or more of the functions of the
electronic circuit 130. In other words, various functions, such as
sensing, amplifying, controlling, filtering, reading out, etc.
which can be implemented on the separate electronic circuit 130 can
be implemented, alternatively, on the integrated circuit 150. The
integrated circuit can be formed on a separate layer or wafer and
subsequently connected to the sense layer having the sensor
electrode thereon. The inventors envisage, however, that the
integrated circuit 150 is fabricated within the sense layer
together with the sensor electrode. An integrated circuit can be
provided for each nanopore sensor 102.
[0199] According to some embodiments, after fabrication of the
sense layer 144 having the integrated circuit 150 and sensor
electrode 126 formed and exposed on one side, the sensing structure
is flipped and bonded to the base layer in the same way as it was
in relation to FIG. 3(a). Connections 128 (not shown in FIG. 3(b)
connect the integrated circuit with a connector for sending signals
or data produced by the integrated circuit off the structure. The
connections can be connected to a connector 151 as shown in FIG.
4(e), although other configurations are implementable. With the
sense layer connected to the base layer the nanopore layer 110 can
be formed thereon such that the sense layer is sandwiched between
the nanopore layer and the base layer. As before with FIG. 3(a),
when bonded together, the passages of the sense layer 144 align
with the channels of the base layer and the well of the nanopore
layer form a portion of the passage 114.
[0200] In use, the electronic circuit and/or integrated circuit 150
is configured to detect resistance changes at the nanopore when an
analyte, such as a polymer, passes through the nanopore, said
resistance change detected through the fluid in the sensor (e.g., a
measure of resistance being detected as a voltage over the
effective voltage divider, as described above). For example,
changes in resistance at the nanopore can cause changes in an
applied voltage, which is detected by the circuit 150. In an array
of nanopore structures 104 the integrated circuit of each sensor
102 can be communicably addressable. In light of parasitics, noise
from communications, and background noise, the detected voltage
changes or the detected resistance can be difficult to read
directly using an off-board processor. To provide a processor with
a better signal, i.e. a cleaner reduced noise signal, the
integrated circuit can be configured to locally transform or modify
or otherwise process signals derived from the detection of a
polynucleotide or other analyte passing through the nanopore 116.
In some embodiments, the integrated circuit can be configured to at
least one of: amplify signals, such as amplifying a voltage level
of the signal; filter the signal, for example to remove noise;
sample the signal; digitise the signal using an analogue to digital
converter (ADC) implemented in the electronic circuit.
[0201] According to some embodiments, at least one integrated
circuit 150 can be formed or packaged within at least one nanopore
sensor 102 footprint within the array of nanopore structures 104 of
the structure 100.
[0202] By way of example, each nanopore sensor 102 of the array of
nanopore structures 104 can be contained or packaged within a
nanopore sensor footprint 101, which can be regarded as a footprint
of a nanopore sensor 102, as viewed in FIG. 4(a), which can be
considered to represent a schematic plan view of a nanopore sensor
102 depicted in FIG. 3(b). As illustrated in FIG. 4(a), each
nanopore sensor footprint 101 accommodates a nanopore sensor 102,
an sensor electrode 126, and integrated circuit 150. The sensor
electrode 126 and integrated circuit can be arranged to inhibit
noise interference created by the integrated circuit from being
detected by the sensor electrode 126. For example, the integrated
circuit 150 may be separated from the nanopore sensor 102, as
depicted in FIG. 4(a). This separation can be implemented by
configuring the integrated circuit 150 to be located outside the
nanopore sensor footprint 101, as viewed.
[0203] This separation may simplify (e.g., simplifies) the
manufacturing process. Alternatively, the integrated circuit 150
can be distanced from the sensor electrode (e.g., the distance
between the electrode and the circuit in the depth direction, or
thickness of the structure, and/or lateral distance is increased to
minimise noise interference). Note that the depth direction of FIG.
4(a) is in a direction into and out of the page, as viewed.
[0204] In the example shown, the nanopore sensor footprint 101 is
square and has a side length of 20 .mu.m, but in other examples may
be in a range from 10 .mu.m to 50 .mu.m. By way of example, the
integrated circuit occupies about three-quarters of the footprint,
while the remaining quarter is occupied by the sensor electrode 126
which has a diameter of 10 .mu.m in the example shown.
[0205] Other arrangements are envisaged. In some embodiments, the
sensor electrode 126 may be larger than the example shown in FIG.
4(a), for example covering almost all of the area of the nanopore
sensor. In some embodiments, the sensor electrode 126 may be have
other shapes covering more area, for example square or rectangular.
The sensor electrode 126 may have dimensions of up to 50 .mu.m, in
which case it may have an area of up to 250 .mu.m.sup.2, depending
on its shape. In some embodiments, pixels may be square or
rectangular with a larges edge dimension between 5 microns and 60
microns.
[0206] For packaging efficiency, the pixel can be tessellated, and,
for example, the tessellation can be hexagonal.
[0207] Each sensor 102 has a passage 114, although during
fabrication of the base layer 112 more channels 122 could be
created in the base layer 112 than are needed, depending on the
method of fabrication. Some methods of fabrication such as reactive
ion etching can etch a single channel for each footprint 101. Some
other method such as photo assisted electrochemical etching
requires a high-density array of channels to be etched at the same
time to maintain the geometry of those channels--in this case
unused channels in the base layer are blocked during fabrication of
the array of nanopore structures 104 such that only one channel and
one passage are provided per nanopore sensor footprint 101. The
density of the channels 122 formed in the base layer 112 can vary.
FIG. 4(b) shows, by way of comparison, a cross-section of a
nanopore sensor having a lower density of blocked channels 122a
than that shown in FIG. 4(a). The channels, as shown in FIG. 4(b)
can be blocked prior to the sense layer 144 being added to the base
layer, or may be blocked by a substrate of the sense layer. It is
to be noted that FIG. 4(b) is shown with portions of two nanopore
structures, each with its own passage 114, and has not yet had a
nanopore layer 110 added upon the sense layer 144.
[0208] FIG. 4(c) shows the nanopore sensor 102 footprint 101 of
FIG. 4(a) arranged in a 6.times.6 layout providing an array of
nanopore structures 104 of 36 nanopore sensors, while FIG. 4(d) has
an 18.times.18 array having 324 nanopore sensors. The array size
can be 1000.times.1000, providing 1,000,000 nanopore sensors. In
the present example, an array of one million sensors of the type
shown in FIG. 4(a) would have a footprint of 4 cm.sup.2, however
sensors having pixels as small as 5 .mu.m can bring the footprint
of a one million sensor array down to around 25 mm.sup.2. The array
size can be 100,000. The array may comprise any number of sensors
between 1000 and 10 million sensors.
[0209] FIG. 4(e) shows an array of nanopore structures 104 having
nanopore sensors 102 as described herein arranged in a structure
100 provided in a device 149 for receiving and analysing an analyte
of polymer such as nucleic acid. The array of nanopore structures
104 can be a sub-component of the device. The array can be a
disposable component and replaceable. Additionally, or
alternatively, the nanopore layer 110 of the array of nanopore
structures 104 can be a disposable component and replaceable. While
some of the inventive aspects relate to a device as a whole, some
inventive aspects can also reside in the nanopore sensor 102 and/or
the array of nanopore structures 104. The device 149 can include a
connectable circuit 130 as described above.
[0210] In some embodiments, processing of the signals measured by a
nanopore sensor can be performed by the circuit 130. In some
embodiments, the integrated circuit 150 can perform pre-processing
prior to further analysis by the circuit 130 of the device 149.
[0211] According to some embodiments, the device 149 houses the
structure 100 to separate and define the analyte chamber 106 and
outlet chamber 108. While often referred to, respectively, as the
cis and the trans, the analyte can flow from the analyte chamber to
the outlet chamber. The array of nanopore structures 104 has a
plurality of nanopore sensors 102, each with a passage
therethrough, to fluidly connect the cis and trans. By way of
example, the electrodes 132 in the cis and trans can impose an
electrical potential difference across the fluidic passage, between
the first and second fluidic reservoirs, to induce an analyte to
flow from the cis to the trans. The electrodes can be configured
such that the potential difference is substantially the same across
all the nanopore sensors 102.
[0212] Additionally, or alternatively, the device can be configured
to induce an analyte from the cis to the trans using other
techniques. As an analyte passes through a nanopore the fluctuation
in electrical potential caused by changes in ion current flow is
detected by the sensor electrode 126.
[0213] The sensor electrode 126 can function as, or connect
directly to, the base of a transistor, which can be a gate of a
field effect transistor (FET) device, for example. The transistor
outputs a signal that can be processed by the integrated circuit
150 of each sensor 102, which can then be addressed in a row-column
type manner. For example, the voltage at the drain of the
transistor may depend upon the electrical potential sensed by the
sensor electrode 126, and the voltage at the drain can be read out,
along with other drain voltages on other nanopore sensors 102 in an
array of nanopore structures 104, in a row-column manner. The
processed signal(s) can then be analysed further--off the array of
nanopore structures 104--to determine one or more properties of the
analyte.
[0214] In the examples above, each nanopore sensor footprint 101
has its own integrated structure 150, but an integrated structure
can be configured to serve a plurality of nanopore sensors. In FIG.
4(f), four nanopore sensors 102 are shown as a sensor module 102a,
wherein the integrated circuit 150 is common to four centrally
located electrodes, as shown. Other configurations are feasible. In
such module configurations the information or data obtained from
each individual sensor is addressable for control and/or retrieval
of information. While the examples above have a dedicated
integrated circuit for each nanopore sensor 102, combining nanopore
sensors into a module enables the efficiency of the layout to be
improved. Efficiency improvements can, for example, be achieved
because a common filter is used for each of the nanopores within
the module. This is possible if the integrated circuit switches or
multiplexes between the individual nanopore sensors in turn. By
sharing functions between the nanopore sensors either the footprint
of the integrated circuit can be reduced or, alternatively, more
functions can be accommodated.
[0215] FIG. 5(a) is an example schematic representation of the
connections to the sensor electrode 126 for each sensor 102 in the
array of nanopore structures 104. In some embodiments, the cis
electrode 132a can be connected to ground while a translocation
voltage is applied to trans electrode 132b, though other
configurations of applied voltage(s) and/or grounding may be used.
The resistance of the nanopore 116 and the resistance of the
channel 122, which is configured to function as a fluidic resistor,
dominate the circuit between the electrodes via each passage 114 of
each sensor 102. In this way the circuit behaves like a voltage
divider having two resistors of similar value. The nanopore
resistance and resistance of the channel or fluidic resistor, are
approximately the same such that an electrode positioned
therebetween is positioned to detect changes in the nanopore
resistance caused by an analyte passing therethrough. The sensor
electrode 126 resides, as described above, in the region of each
nanopore. The sensor electrode 126 can lie between the nanopore and
the channel. The effective impedance of the nanopore and the
channel are much larger than the bulk fluidic resistance of the cis
reservoir and trans reservoir--this means that FIG. 5(a) can be
used to model the circuit between the electrodes.
[0216] The circuitry includes a sensing circuit 152 which is
configured to detect changes in fluidic electrical potential at the
sensor electrode 126 of the nanopore structure for producing
signals from the nanopore that are indicative of analyte
characteristics.
[0217] The sensing circuit 152 may include, for example a sensing
transistor 153 which may be a field effect transistor (FET). In
this case, the electrode 126 may be connected to the base of the
sensing transistor 153. The sensing circuit 152 may reside, at
least in part, in the integrated circuit 150. Thus, the sensor
electrode 126 may be connected to a sensor terminal 154 of the
sensing circuitry 152, as shown in FIG. 5(a).
[0218] In some cases, the sensor electrode 126 can additionally be
connectable to a control circuit 155, as shown, which applies a
signal to the sensor electrode to alter an electrical potential
difference across the nanopore imposed by the drive electrodes 132
in response to a control signal. The control circuit 155 may
include, for example a control transistor 156 which may be a field
effect transistor (FET). In this case, the electrode 126 may be
connected to the drain of the control transistor 156. The sensing
circuitry and/or control circuitry 155 can reside, at least in
part, in the integrated circuit 150. Thus, the sensor electrode 126
may be connected to a control transistor 156 of the control circuit
155, as shown in FIG. 5(a) for application of the control
signal.
[0219] The application of the control signal enables an alteration
of the potential difference imposed across the individual nanopore
by altering the potential difference between the control connection
of the control circuit 155 and the analyte electrode and/or the
outlet electrode. The signal applied to the sensor electrode can be
a reverse-voltage that induces the charged analyte, such as a
species, to change the direction in which it is moving through the
passage 114.
[0220] In some cases, the voltage applied can be an alternating
current voltage, though other voltage waveforms (e.g., ramp, step,
impulse, DC) may be applied.
[0221] The circuit of FIG. 5(a) enables a common electrode to be
configured for each of the cis and trans reservoirs, while each
nanopore sensor 102 can operate to detect an interruption to ion
current flow across the passage by detecting variations in
electrical potential caused by a variation in nanopore resistance.
Furthermore, the circuit enables each nanopore sensor 102 within
the array of nanopore structures 104 to be individually controlled
to enable the sensor electrode to either detect an analyte passing
through the pore through, for example, a connection with a sensing
FET or control the flow of a charged analyte, such as a species, in
the passage 114 of individual sensors 102 in the array of nanopore
structures 104 by adjusting the voltage applied to the sensor
electrode 126 using, for example, a control FET. The control of the
flow of a charged analyte, such as a species, in the passage 114 of
individual sensors 102 in the array allows for an analyte passing
through the nanopore 116, or an analyte blocking the nanopore, to
be passed back or ejected by a voltage applied by the control FET.
This action can be described as "flicking" or "rejecting" and
occurs by using a control voltage, such that an analyte passing
from one side of the structure 100 through the passage 114 is
modified--either stopped, reversed or accelerated. A control
voltage can be applied to each pore, individually, because each
sensor 102 is individually addressable for controlling and sensing.
To be clear, the application of a control signal to the electrode
126 in each sensor 102 means that the voltage near the nanopore 116
at each pixel can be controlled.
[0222] The control voltage can be applied to alter the movement of
an analyte through the nanopore 116 in response to at least one
condition from conditions including: when a blocked pore is
detected; when the analyte detected is no longer of interest and is
to be ejected for the purposes of enabling another sample to be
received and measured; and to alter the rate at which an analyte is
induced into or out of the pore.
[0223] An electronic sensor, inevitably, has capacitances,
resistances and inductances associated with the path along which
the sensor signal travels, which may be referred to as parasitics.
These are due to the properties of the materials the sensor is
constructed from, the geometry of the sensor, and the methods by
which it is feasible to fabricate the sensor. Without any kind of
capacitance compensation, these parasitics (most commonly the
resistances and capacitances) interact to limit the bandwidth of
the signal. In the simplest case, a resistor-capacitor circuit will
limit the bandwidth to 1/(2 .pi. R C).
[0224] FIG. 5(b) is an alternative schematic of FIG. 5(a) that
illustrates the resister model 161 of the nanopore 116 and the
channel 122 and further includes a compensation circuit 160
connected to the voltage divider. According to some embodiments, a
compensation circuit 160 has an inline amplifier 168, with gain G,
connected to the output of the sensor electrode 126, which is
influenced by the parasitic input capacitance 162. The output of
the inline amplifier has a feedback loop connected to its input,
said feedback loop having a feedback amplifier 170, with gain H,
and a capacitor C representing compensation capacitance
C.sub.compensation.
[0225] A capacitor 162 is shown arranged in parallel with the
resistor representing the channel 122, which represents parasitic
capacitance in at least one of: the membrane in which the nanopore
rests; the fluidic walls of the channel; the electrode; and a trace
capacitance associated with a connector or wire-bond. The sensor
electrode 126 is, in effect, connected to the mid-point in the
voltage divider between the nanopore and channel and connected to
the compensation circuit 160. The connection to a reverse or
flicking voltage is represented by a flicking switch 164, such as a
FET. An optional guard switch 166 is shown implemented between the
sensor 126 and the compensation circuit. This switch, which can be
implemented using a FET, can function to isolate the compensation
circuit 160 and/or any sensing circuitry connected thereto from the
flicking voltage applied via the flicking switch 164.
[0226] Overall, the compensation circuit 160 mitigates the effects
of the total parasitic capacitance 162 at the input to the sensing
stage. Although the parasitic capacitance resides in various
elements of the sensor 102 it can be modelled as shown in FIG.
5(b). Without being bound to a particular theory, a total parasitic
capacitance 162 C.sub.p can be considered as a sum of various
parasitic capacitances, as follows.
C.sub.p=C.sub.membrane+C.sub.fluidic
walls+C.sub.electrode+C.sub.trace
[0227] The rate at which the input capacitance charges is
proportional to the current flowing through it. In turn, the
resistance limits the charging current to a finite value. The
compensation circuit 160 functions to supply additional current to
charge the input capacitance faster, thus increasing the
bandwidth.
[0228] In some embodiments, the input voltage is amplified and fed
back through the compensation capacitor so as to provide additional
current to charge the total parasitic capacitance 162. According to
some embodiments, an effective input capacitance of this circuit
can be expressed as:
C.sub.effective=C.sub.p-C.sub.compensation
wherein
C.sub.compensation=C*(G*H-.sup.1)
[0229] The components of the compensation circuit 160 are
configured such that the total parasitic capacitance C.sub.p is
substantially negated or cancelled. In practice, the degree of
compensation is limited by dynamic changes in the component values
and parameters (e.g. temperature dependence). The compensation
circuit can compensate for a range of different parasitic
capacitance values if capacitance C, inline gain G or feedback gain
H is made adjustable, hence the feedback amplifier is illustrated
as variable in FIG. 5(b). The gain G can be fixed such that the
output from the compensation circuit has a consistent gain,
therefore either the capacitor C and/or the feedback gain H can be
varied.
[0230] The front-end electronics can reside, at least in part, in
the integrated circuit 150, which is figuratively represented in
FIG. 5(c). In some embodiments, the control circuit 155 and/or
compensation circuit 160 can optionally be incorporated within the
integrated circuit 150. The integrated circuit or electronic
circuit is operable to influence the movement of an analyte in the
nanopore, such as by flicking, by applying a reverse voltage, and
amplifying the signal from the nanopore sensor 102. The integrated
circuit 150 or electronic circuit 130 can additionally incorporate
further processing of the signal, such as filtering, and may
include circuitry to store information locally at the sensor 102,
in the case of the integrated circuit, for managed communication
with an external processor.
[0231] According to some embodiments, each nanopore sensor 102,
such as those illustrated in FIG. 2 and FIG. 3(a), can be
addressable. FIG. 5(c) represents the nanopore sensor 102 of FIG.
3(b) that incorporates the integrated circuit 150 within a nanopore
sensor footprint 101, as shown in FIG. 4(a) for example, and is
also addressable via row-selection and column bus. FIG. 5(d) is an
example of a row-column readout circuit 174 connected to each
sensor 102 in an array of nanopore structures 104, such as the
array shown in FIG. 4(d), via a row-selection and column bus
connection shown in FIG. 5(c). Each sensor 102 is connected to a
row decoder 176 and column readout 178 via an analogue to digital
converter 180. The readout circuit 174 can be connected to the
integrated circuit 150 of each sensor 102 or group of sensors in
some embodiments, or may be connected directly to the sensor
electrode 126 in each nanopore sensor 102 within the array of
nanopore structures 104.
[0232] The examples above describe the sensor electrode 126 being
connectable to an integrated circuit 150 and having the option of
dual functionality when a control voltage is applied (i.e. the
sensor electrode 126 can be used to sense the change of ion flow
when an analyte passes through a nanopore and create an electrical
potential within the passage and a potential difference across the
passage between the cis and or trans electrodes 132 under the
control of the control circuit 155). In this case, the sensor
electrode 126 is directly connected to a control terminal, which is
a terminal of the integrated circuit 150, for creating a potential
difference across the passage. In some embodiments, the sensor
electrode 126 can be connected to a control terminal, which is a
terminal of the integrated circuit 150, for creating a potential
difference across the passage.
[0233] In some implementations, the sensing and control functions
in each sensor 102 can be implemented by separate electrodes. FIG.
6(a) shows a sensor electrode 126 and control electrode 182
arranged like an annulus, while FIGS. 6(b) to 6(e) are
cross-sectional schematics of nanopore sensors 102 having
configurations in which a control electrode 182 is provided in
addition to the sensor electrode 126. In this case, the control
electrode 182 is connected to a control terminal of the control
circuit 155 for creating the electrical potential within the
passage. In some embodiments, the control electrode 182 can be
connected to a sensing circuit but additionally or alternatively
can be connected to a control terminal of the control circuit 155
for creating the electrical potential within the passage.
[0234] In an example herein, the sensor electrode 126 has been
described as an annulus, as illustrated in FIG. 4(a). The sensor
electrode could also be implemented by an exposed wire. The sensor
electrode could be a nanowire, but can be a larger surface area
that occupies, for example, substantially all of the base of a well
142, as shown in FIG. 6(e), or one face of a recess 146. Similarly,
a separate control electrode 182 could be a nanowire but can have a
large surface area, as shown in FIG. 6(d).
[0235] From a manufacturability and cost perspective, a basic
implementation of a control electrode 182 is shown in FIG. 6(a),
wherein the annulus footprint--suitable for the base of a well
142--is substantially maintained, while one half of the footprint
forms the sensor electrode 126 and the other half, which is
physically disconnected or decoupled from sensor side, forms the
control electrode 182. There is no wired or solid-state connection
between the sensor electrode 126 and the control electrode 182. The
electrodes 126, 182 are shown having two equally sized semi-circle
shapes occupying the footprint. In some embodiments, the electrodes
can be different sizes, and, for example, the control electrode can
have a greater surface area than the sensor electrode to increase
the conductivity with the fluid within the passage.
[0236] Having separate sensor and control electrodes can simplify
the integrated circuit because, by being separate, an extra degree
of separation is provided, although they will still be connected
through a fluid in the passage. However, it can be possible to
avoid the need of isolating switch to protect, for example, the
compensation circuit 160, which can form part of the sensing
circuit, from the voltages applied by the control circuit. The
electrodes can be tailored in shape, size and configuration to be
optimised for their purpose.
[0237] FIG. 6(b) indicates how the electrode of FIG. 3(b) can be
divided into separate sensor electrode 126 and control electrode
182. In this example the electrodes extend in the same plane. In an
alternative configuration shown in FIG. 6(c) the sense electrodes
reside in the cavity 146 and extend in a plane extending in
parallel with the cis-surface 134 and trans-surface 138, while the
control electrodes extend in the channel 122 and extend
perpendicularly from said surfaces. In FIG. 6(c) the sensor
electrode 126 is shaped like an annulus while the control electrode
is shaped like a cylinder. In yet another alternative, as shown in
FIG. 6(d), the sense electrodes reside in the cavity 146 and extend
in a plane extending in parallel with the cis-surface 134 and
trans-surface 138, while the control electrodes extend in the
channel 122 and in the cavity 146, thus extending in vertical and
horizontal planes, as viewed. FIG. 6(e), which is similar to FIG.
6(b), shows the electrodes 126, 182 formed at the base of the well
142, which can offer easier fabrication.
[0238] As described above, an electronic sensor inevitably has
capacitances, resistances and inductances associated with the path
along which the sensor signal travels, which may be referred to as
parasitics, which includes parasitic capacitances. Additionally, or
alternatively to the compensation circuit 160 described above, the
array of nanopore structures 104 and sensors therein can be
fabricated with a guard conductor 184 incorporated therein, as
shown in FIGS. 7(b) to 7(d), while FIG. 7(a) shows first and second
schematic circuits 201, 202 with and without a guard conductor 184
in order to illustrate how a guard conductor 184 is configured. In
the left-hand first schematic circuit 201 of FIG. 7(a), the
parasitic capacitance C.sub.parasitic is shown between two
conductive elements 203, 204 of the sensor 102, typically being a
conductor, such as the sensor electrode 126 and a conductive
substrate of the base layer. The first conductive element 203 (e.g.
sensor electrode 126) may carry a voltage V.sub.sensor and the
second conductive element 204 (e.g. the conductive substrate) may
carry a different voltage V.sub.substrate.
[0239] Guarding is shown implemented in the right-hand, second
schematic circuit 202, wherein the guard conductor 184 which is a
third conductive element is configured between the first conductive
element 203 carrying the signal and the second conductive element
204 such that two parasitic capacitances C.sub.par1, C.sub.par2 can
be modelled as connected in series. In this schematic 202, the
parasitic capacitance occurs (i) between the first conductive
element 203 or conductor carrying the signal and the guard
conductor 184 (i.e. C.sub.par1) and (ii) between the guard
conductor 184 and the second conductive element 204 (i.e.
C.sub.par2). According to some embodiments, a buffer 205 (which may
be an amplifier) is connected between the first conductive element
203 and the guard conductor 184 to apply a buffered version of the
input signal is applied to the guard conductor 184. As a result,
there is no voltage difference across the parasitic capacitance
C.sub.par1 between the first conductive element 203 and the guard
conductor 184.
[0240] For a capacitor, the current is given by
I = C d V d t ##EQU00001##
[0241] In the right-hand schematic, V.sub.guard=V.sub.sensor,
thus
d V d t = 0 . ##EQU00002##
[0242] No current flows through the capacitor C.sub.par1, thus the
effective capacitance is zero. The capacitance between the guard
and the substrate conductors must still be charged, but the buffer
205 is able to supply much more current than the high-impedance
sensor input, so it charges much faster.
[0243] These conditions are met when V.sub.guard accurately follows
V.sub.sensor, which depends on the performance of the buffer 205
having sufficient bandwidth to enable the capacitance to be nulled.
Precise buffers with bandwidths of several MHz can be
implemented.
[0244] FIG. 7(b) is analogous to FIG. 2(b) and shows, by way of
comparison, a guard conductor 184 extending between oxide layers
192 along the length of the channel 122, vertically as viewed, and
continues horizontally, as viewed, along the top of the base layer
112 beneath the sensor electrode 126. Notably, both the sensor
electrode 126 and the guard conductor 184 are connected to the
separate electronic circuit 130. In this configuration the guard
conductor inhibits current flow in the parasitic capacitance
between the sensor electrode and the substrate of the base layer.
The conductive guard can include, at least in part, a guard
conductor and an insulating layer that insulates the guard
conductor from the conductor being guarded or the conductor being
guarded from. The insulating layer is not part of the guard and
functions to isolate the guard from surrounding conductors. The
insulating layer, therefore, can be a non-conductive component of
the structure 100. The guard can be a conductor inserted into the
middle of the parasitic capacitances to divide them in two, which
is possible because capacitors are by nature insulators, thus the
guard is located in an existing insulating layer.
[0245] The guard conductor 184, including an insulating layer, can
be configured in a number of different configurations, or
combination thereof, comprising at least one of: extending over at
least a portion of the nanopore layer 110 for separating the
nanopore layer from an analyte in the analyte chamber 106, as shown
in FIG. 7(c), which guards the solution underneath the nanopore
from the solution above; extending between the at least a portion
of the nanopore layer 110 and the sense layer 144 for separating
the sensor electrode 126 and integrated circuit 150 from the
solution in the cis 106, as also shown in FIG. 7(c); extending
between the base layer 112 and the sense layer 144, at least in
part, for separating the sensor electrode 126 and integrated
circuit 150 from the base layer, as also shown in FIG. 7(c); and a
plurality of conductive guards, as shown in FIG. 7(d), wherein a
first guard extends between the walls of the channel 122 and the
base layer 112 and a second guard extends between the sense layer
144 and the base layer.
[0246] In light of the teaching herein a skilled person would
appreciate that one of the guard arrangements taught herein, or a
combination thereof, could be implemented. It will also be
appreciated that the guard conductor 184 can be provided in an
array of nanopore structures, e.g. the array of FIG. 4(c).
[0247] It is to be noted that guard-based capacitance compensation
techniques, shown in FIGS. 7(a) to 7(d) have the advantage that
they generally do not appreciably increase the noise level of the
signal. However, such a technique cannot compensate for the
membrane capacitance when a potential difference across the
membrane is used to drive the analyte being studied through the
pore, but it may be possible to drive the analyte by another means,
e.g. pressure. A compensation circuit 160, on the other hand, can
compensate for the entire input capacitance, but does so at the
expense of added noise. The noise gain of the feedback capacitor
172 increases with frequency. Therefore, the noise in the input
signal is scaled by this feedback gain `G` and adds to the overall
noise. This becomes significant at higher frequencies or when
compensating for larger input capacitances. The guard conductors
shown in FIGS. 7(b) to (d) can, therefore, be implemented in the
array of nanopore structures 104 in any combination and/or in
combination with a compensation circuit 160.
[0248] The sensors 102 can be manufactured using a number of
different techniques and the functions are taught, by way of
example with reference to FIG. 2, which is indicative of the other
sensors taught in the application. Although only one of the sensors
102 of the array of nanopore structures 104 is shown in FIG. 2, the
fabrication of an array can be understood from the teaching herein.
The base layer 112 is formed from a standard silicon (Si) wafer
that has channels 122 formed therein to pass from one side of the
layer to the other. Only one channel is shown in FIG. 2, formed
through the Si wafer extending substantially perpendicularly to the
surfaces of the wafer. In practice, the array has channels formed
across the wafer using techniques such as photolithography or deep
reactive-ion etching (DRIE) or combinations thereof. At least one
channel is formed for each sensor. Techniques such as thermal
oxidation can be used to adjust the diameter of the channel to
calibrate the aspect ratio, if required. In some cases, the
channels can be embedded in an oxide layer, which may be formed on
a silicon wafer, for example.
[0249] The example of FIG. 2 schematically shows a portion of the
structure 100 having an array of nanopore structures 104 of
nanopore sensors 102 (only one of which is shown) and is configured
to separate a cis and trans having electrodes 132 therein. All of
the sensors 102 herein can be located in a structure as shown in
FIG. 4(e). The nanopore 116 lies in the passage 114 between the cis
and the trans, which are fluid filled. The passage is fluid filled
such that the cis and trans are fluidically connected. To be clear,
the nanopore lies in a path of fluidic communication between the
analyte chamber 106 and outlet chamber 108.
[0250] FIGS. 8 and 9 show two further examples of a device 149
including incorporating a structure 100. In each case, the
structure 100 takes the form shown in either FIG. 3(a) or 3(b)
including a nanopore layer 110, a sense layer 144 and a base layer
112, as described in detail above (although in each case it could
be replaced by a structure 100 taking the form shown in FIG.
2).
[0251] In each of the examples of FIGS. 8 and 9, the structure 100
separates the analyte chamber 106 and the outlet chamber 108 and is
connected to a printed circuit board 210 but with different
configurations as follows.
[0252] In the example of FIG. 8, the analyte chamber 106 and the
outlet chamber 108 are each formed by respective gaskets 216, 218
which seal against the nanopore layer 110 and the base layer 112,
respectively. The analyte chamber 106 and the outlet chamber 108
may be open as shown in FIG. 8 or may be closed, for example by
respective members extending across the gaskets 216, 218.
[0253] In the example of FIG. 8, the printed circuit board 210 is
mounted to the base layer 112 by a mechanical bond 212 (e.g.
adhesive) on the opposite side from the nanopore layer 110. Thus,
the printed circuit board 210 is disposed outside the outlet
chamber 108, as shown in FIG. 8. The sense layer 144 is connected
to the printed circuit board 210 by a wire bond 214, or any other
suitable electrical connection. The nanopore layer 110 has a
smaller area than the sense layer 144 to provide space for the wire
bond 214.
[0254] In the example of FIG. 9, the printed circuit board 210 is
mounted to the sense layer 144 by a solder bump connection 222
(e.g. adhesive) on the same side as the nanopore layer 110. Thus
the nanopore layer 110 has a smaller area than the sense layer 144
to provide space for the solder bump connection 222. The solder
bump connection 222 provides both mechanical and electrical
connection between the printed circuit board 210 and the sense
layer 144.
[0255] In the example of FIG. 9, the analyte chamber 106 and the
outlet chamber 108 are each formed in respective flowcells 224, 226
which may be made of any suitable material, for example plastic.
The flowcells 224, 226 allow flow of fluid into and out of the
analyte chamber 106 and the outlet chamber 108.
[0256] The flowcell 224 that forms the analyte chamber 106 is
sealed to the printed circuit board 210 around the analyte chamber
106 by a gasket 228, and the printed circuit board 210 is sealed to
the edges of the nanopore layer 110 around the analyte chamber 106
by a sealant 230.
[0257] The flowcell 224 that forms the outlet chamber 108 is sealed
to the base layer 112 around the outlet chamber 108 by a gasket
232.
[0258] The examples of FIGS. 8 and 9 can be modified in various
ways, for example to provide sealing in other locations (e.g.
around the outside edge of the base layer 112) and by any suitable
means.
[0259] The electrical model of a nanopore sensor has been described
above. More generally, a voltage source, not shown in FIG. 2,
applies a potential difference between the electrodes configured in
the chambers 106, 108. The electrodes impose an electrical
potential across the passage 114, including the nanopore 116 and
channel 120. The nanopore resistance and channel resistance are
significantly higher than the overall fluidic resistance of the
reservoirs and, therefore, the nanopore and channel are the
dominant components in an equivalent electrical circuit. As shown
in FIG. 2, the sensor electrode 126 lies between the nanopore and
channel such that it can sense the fluidic electrical potential at
the sensor electrode in the passage 114. In other words, the sensor
electrode can sense a signal indicative of local electrical
potential fluctuations in the passage. Although the configuration
in FIG. 2 is an example, the sensor electrode 126 can be located in
the cis 106 or trans 108. The sensor electrode 126 can function as
the base or gate of a transistor device for measuring electrical
potential of the fluid at the location of the sensor electrode 126
when a fluid is provided in the passage. The sensor electrode 126
can detect fluctuations in voltage as a species object, such as a
strand of DNA, translocates through the nanopore 126.
[0260] The embodiments herein have described a device having a
single sample reservoir separated from a single outlet chamber by
the structure 100. In light of the teaching herein alternative
arrangements can be implemented and include a device having (i) two
or more sample reservoirs separated from a common outlet chamber by
the structure, (ii) a common sample reservoir separated from two or
more outlet chambers by the structure, or (iii) two or more sample
reservoirs separated from two or more respective outlet chambers by
the structure.
[0261] The nanopore layer 110 can be formed separately having an
array of wells that can be formed in a number of ways, one of which
is by lithographically patterning a polymer layer. The wells in the
nanopore layer are then aligned with the channels of the base layer
such that each sensor 102 has a passage 114 defined by the well 142
and channel 122. The well 142 shown in FIG. 2 is substantial in
comparison to the nanopore 116 located in the membrane 118. The
nanopore of FIG. 2 is a biological nanopore in a membrane such as
an amphiphilic membrane. Alternatively, each nanopore can be a
solid state nanopore located in a solid-state membrane. The
solid-state membrane itself can be the nanopore layer 110. Further
alternatively, the nanopores can be biological nanopores located in
a solid-state membrane. In light of the dimensions of the nanopore
relative to the width of the channel, which is greater in diameter,
a well can be said to form beneath the nanopore. The nanopore,
therefore, defines a part of the passage 114 in each of the
alternative nanopore configurations.
[0262] Any membrane may be used in accordance with various aspects
described herein. An example membrane can comprise an amphiphilic
layer or a solid-state layer. An amphiphilic layer is a layer
formed from amphiphilic molecules, such as phospholipids, which
have both hydrophilic and lipophilic properties. The amphiphilic
molecules may be synthetic or naturally occurring. Non-naturally
occurring amphiphiles and amphiphiles which form a monolayer
include, for example, block copolymers (Gonzalez-Perez et al.,
Langmuir, 2009, 25, 10447-10450). The copolymer may be a triblock,
tetrablock or pentablock copolymer. The membrane can be a triblock
or diblock copolymer membrane.
[0263] Membranes formed from block copolymers hold several
advantages over biological lipid membranes. Because the triblock
copolymer is synthesized, the exact construction can be carefully
controlled to provide the correct chain lengths and properties
required to form membranes and to interact with pores and other
proteins.
[0264] Block copolymers may also be constructed from sub-units that
are not classed as lipid sub-materials; for example, a hydrophobic
polymer may be made from siloxane or other non-hydrocarbon-based
monomers. The hydrophilic sub-section of block copolymer can also
possess low protein binding properties, which allows the creation
of a membrane that is highly resistant when exposed to raw
biological samples. This head group unit may also be derived from
non-classical lipid head-groups.
[0265] Triblock copolymer membranes also have increased mechanical
and environmental stability compared with biological lipid
membranes, for example a much higher operational temperature or pH
range. The synthetic nature of the block copolymers provides a
platform to customize polymer-based membranes for a wide range of
applications.
[0266] The membrane can be one of the membranes disclosed in
WO2014/064443 or WO2014/064444, each of which is hereby
incorporated by reference in its entirety. These documents also
disclose suitable polymers.
[0267] The amphiphilic molecules may be chemically-modified or
functionalized to facilitate coupling of the polynucleotide.
[0268] The amphiphilic layer may be a monolayer or a bilayer. The
amphiphilic layer can be planar (e.g., is planar). The amphiphilic
layer may be curved. The amphiphilic layer may be supported. The
amphiphilic layer may be concave. The amphiphilic layer may be
suspended from raised pillars such that the peripheral region of
the amphiphilic layer (which is attached to the pillars) is higher
than the amphiphilic layer region. This may allow the microparticle
to travel, move, slide or roll along the membrane as described
above.
[0269] The membrane may be a lipid bilayer. Suitable lipid bilayers
are disclosed in WO 2008/102121, WO 2009/077734 and WO
2006/100484.
[0270] Various methods for forming lipid bilayers may be used. For
example, lipid bilayers can be formed by the method of Montal and
Mueller (Proc. Natl. Acad. Sci. USA., 1972; 69: 3561-3566), in
which a lipid monolayer is carried on aqueous solution/air
interface past either side of an aperture which is perpendicular to
that interface.
[0271] Solid state layers can be formed from both organic and
inorganic materials including, but not limited to, microelectronic
materials, insulating materials such as Si.sub.3N.sub.4,
Al.sub.2O.sub.3, and SiO, organic and inorganic polymers such as
polyamide, plastics such as Teflon.RTM. or elastomers such as
two-component addition-cure silicone rubber, and glasses. The
solid-state layer may be formed from graphene. Suitable graphene
layers are disclosed in WO 2009/035647. Yusko et al., Nature
Nanotechnology, 2011; 6: 253-260 and US Patent Application No.
2013/0048499 describe the delivery of proteins to transmembrane
pores in solid state layers without the use of microparticles.
[0272] Any transmembrane pore may be used. The pore may be
biological or artificial. Suitable pores include, but are not
limited to, protein pores, polynucleotide pores and solid-state
pores. The pore may be a DNA origami pore (Langecker et al.,
Science, 2012; 338: 932-936).
[0273] The transmembrane pore can be a transmembrane protein pore.
A transmembrane protein pore is a polypeptide or a collection of
polypeptides that permits hydrated ions, such as the by-products of
processing a polynucleotide with a polymerase, to flow from one
side of a membrane to the other side of the membrane. In one
embodiment, the transmembrane protein pore is capable of forming a
pore that permits hydrated ions driven by an applied potential to
flow from one side of the membrane to the other. The transmembrane
protein pore can permit polynucleotides to flow from one side of
the membrane, such as a triblock copolymer membrane, to the other.
The transmembrane protein pore may allow a polynucleotide, such as
DNA or RNA, to be moved through the pore.
[0274] The transmembrane protein pore may be a monomer or an
oligomer. The pore can be made up of several repeating subunits,
such as at least 6, at least 7, at least 8, at least 9, at least
10, at least 11, at least 12, at least 13, at least 14, at least
15, or at least 16 subunits. The pore can be a hexameric,
heptameric, octameric or nonameric pore. The pore may be a
homo-oligomer or a hetero-oligomer.
[0275] According to some embodiments, the transmembrane protein
pore comprises a barrel or channel through which the ions may flow.
The subunits of the pore can surround (e.g., surround) a central
axis and contribute strands to a transmembrane .beta. barrel or
channel or a transmembrane .alpha.-helix bundle or channel. The
barrel or channel of the transmembrane protein pore can include
(e.g., comprises) amino acids that facilitate interaction with
nucleotides, polynucleotides or nucleic acids. These amino acids
can be located near a constriction of the barrel or channel. The
transmembrane protein pore can include (e.g., comprises) one or
more positively charged amino acids, such as arginine, lysine or
histidine, or aromatic amino acids, such as tyrosine or tryptophan.
These amino acids may facilitate (e.g., facilitate) the interaction
between the pore and nucleotides, polynucleotides or nucleic
acids.
[0276] Transmembrane protein pores for use in accordance with the
invention can be derived from .beta.-barrel pores or .alpha.-helix
bundle pores. The transmembrane pore may be derived from or based
on Msp, .alpha.-hemolysin (.alpha.-HL), lysenin, CsgG, ClyA, Sp1
and hemolytic protein fragaceatoxin C (FraC). The transmembrane
protein pore can be derived from CsgG. Suitable pores derived from
CsgG are disclosed in WO 2016/034591. The transmembrane pore may be
derived from lysenin. Suitable pores derived from lysenin are
disclosed in WO 2013/153359.
[0277] The analytes (including, e.g., proteins, peptides, small
molecules, polypeptide, polynucleotides) may be present in an
analyte. The analyte may be any suitable sample. The analyte may be
a biological sample. Any embodiment of the methods described herein
may be carried out in vitro on an analyte obtained from or
extracted from any organism or microorganism. The organism or
microorganism can be (e.g., is) archaean, prokaryotic or eukaryotic
and can belong (e.g., belongs) to one of the five kingdoms:
plantae, animalia, fungi, monera and protista. In some embodiments,
the methods of various aspects described herein may be carried out
in vitro on an analyte obtained from or extracted from any
virus.
[0278] The analyte can be a fluid sample. The analyte can comprise
a body fluid. The body fluid may be obtained from a human or
animal. The human or animal may have, be suspected of having or be
at risk of a disease. The analyte may be urine, lymph, saliva,
mucus, seminal fluid or amniotic fluid, but can be whole blood,
plasma or serum. In some embodiments, the analyte is human in
origin, but alternatively it may be from another mammal such as
from commercially farmed animals such as horses, cattle, sheep or
pigs or may alternatively be pets such as cats or dogs.
Alternatively, an analyte can be of plant origin.
[0279] The analyte may be a non-biological sample. The
non-biological sample can be a fluid sample. An ionic salt such as
potassium chloride may be added to the sample to effect ion flow
through the nanopore.
[0280] The polynucleotide may be single stranded or double
stranded. At least a portion of the polynucleotide may be double
stranded.
[0281] The polynucleotide can be a nucleic acid, such as
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The
polynucleotide can comprise one strand of RNA hybridised to one
strand of DNA. The polynucleotide may be any synthetic nucleic
acid, such as peptide nucleic acid (PNA), glycerol nucleic acid
(GNA), threose nucleic acid (TNA), locked nucleic acid (LNA) or
other synthetic polymers with nucleotide side chains. The
polynucleotide can be any length.
[0282] Any number of polynucleotides can be investigated. For
instance, the method may concern characterising 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 50, 100 or more polynucleotides. If two or more
polynucleotides are characterised, they may be different
polynucleotides or two instances of the same polynucleotide.
[0283] The polynucleotide can be naturally occurring or
artificial.
[0284] The method may involve measuring two, three, four or five or
more characteristics of a polynucleotide. The one or more
characteristics can be selected from (i) the length of the
polynucleotide, (ii) the identity of the polynucleotide, (iii) the
sequence of the polynucleotide, (iv) the secondary structure of the
polynucleotide and (v) whether or not the polynucleotide is
modified.
[0285] For (iii), the sequence of the polynucleotide can be
determined as described previously. Suitable sequencing methods,
particularly those using electrical measurements, are described in
Stoddart D et al., Proc Natl Acad Sci, 12; 106(19):7702-7,
Lieberman K R et al, J Am Chem Soc. 2010; 132(50):17961-72, and
International Application WO 2000/28312.
[0286] The secondary structure may be measured in a variety of
ways. For instance, if the method involves an electrical
measurement, the secondary structure may be measured using a change
in dwell time or a change in ion current flowing through the pore.
This allows regions of single-stranded and double-stranded
polynucleotide to be distinguished.
[0287] The presence or absence of any modification may be measured.
The method can comprises determining whether or not the
polynucleotide is modified by methylation, by oxidation, by damage,
with one or more proteins or with one or more labels, tags or
spacers. Specific modifications will result in specific
interactions with the pore which can be measured using the methods
described below.
[0288] In some embodiments of various aspects described herein, the
method may involve further characterizing the target
polynucleotide. As the target polynucleotide is contacted with the
pore, one or more measurements which are indicative of one or more
characteristics of the target polynucleotide are taken as the
polynucleotide moves with respect to the pore.
[0289] The method may involve determining whether or not the
polynucleotide is modified. The presence or absence of any
modification may be measured. The method can comprises determining
whether or not the polynucleotide is modified by methylation, by
oxidation, by damage, with one or more proteins or with one or more
labels, tags or spacers.
[0290] Also provided is a kit for characterising a target
polynucleotide. The kit comprises a pore as disclosed herein and
the components of a membrane. The membrane can be formed from the
components. The pore can be present in the membrane. The kit may
comprise components of any of the membranes disclosed above, such
as an amphiphilic layer or a triblock copolymer membrane.
[0291] Also provided is an apparatus for characterising a target
analyte, such as a target polynucleotide. The apparatus comprises a
plurality of the pores as disclosed herein and a plurality of
membranes. The plurality of pores can be present in the plurality
of membranes. The number of pores and membranes can be equal. A
single pore can be present in each membrane.
[0292] The apparatus for characterising target analytes, may
comprise or an array of pores as disclosed herein, in a plurality
of membranes.
[0293] The apparatus can further comprises instructions for
carrying out the method. The apparatus may be any conventional
apparatus for analyte analysis, such as an array or a chip. Any of
the embodiments discussed above with reference to the methods are
equally applicable to the apparatus of the invention. The apparatus
may further comprise any of the features present in the kit as
disclosed herein.
[0294] The apparatus can be set up to carry out a method as
disclosed herein.
[0295] The apparatus can comprise: a sensor device that is capable
of supporting the plurality of pores and membranes and being
operable to perform analyte characterisation using the pores and
membranes; and at least one port for delivery of the material for
performing the characterisation. Alternatively, the apparatus can
comprise: a sensor device that is capable of supporting the
plurality of pores and membranes being operable to perform analyte
characterisation using the pores and membranes; and at least one
reservoir for holding material for performing the
characterisation.
[0296] The apparatus can comprise: a sensor device that is capable
of supporting the membrane and plurality of pores and membranes and
being operable to perform analyte characterising using the pores
and membranes; at least one reservoir for holding material for
performing the characterising; a fluidics system configured to
controllably supply material from the at least one reservoir to the
sensor device; and one or more containers for receiving respective
samples, the fluidics system being configured to supply the
analytes selectively from one or more containers to the sensor
device.
[0297] The apparatus may be any of those described in WO
2009/077734, WO 2010/122293, WO 2011/067559 or WO 00/28312.
[0298] Control of the movement of an analyte with respect to the
nanopore e.g. speed of translocation, rejection of the analyte
etc., can be managed by the systems and methods disclosed in
WO2016/059427, incorporated herein by reference in its entirety.
Rejection of an analyte by the nanopore sensor can comprise
ejection of the analyte from the nanopore.
[0299] The features in description above and in Figures of the
invention are interchangeable and compatible in light of the
teaching herein. The present invention has been described above
purely by way of example, and modifications can be made within the
spirit and scope of the invention, which extends to equivalents of
the features described and combinations of one or more features
described herein. The invention also consists in any individual
features described or implicit herein.
LIST OF FEATURES
TABLE-US-00001 [0300] 2 Sensor device 4 Solid-state pore 6 Sample 8
Body 10 Cis 12 Fluidic passage 14 Trans 16 Sensor 18 Electrodes 100
Structure 101 Nanopore sensor footprint 102 Nanopore sensor/pixel
102a Nanopore sensor module 104 Array of nanopore sensors 106
Sample chamber/cis/first fluidic reservoir 108 Outlet
chamber/trans/second fluidic reservoir 110 Nanopore layer 112 Base
layer 114 Passage 116 Nanopore 118 Membrane 120 First end/pore end
122 Channel 124 Second end/channel end 126 sensor electrode 128
Connection/wire-bond 130 Electrical circuit 132 Drive electrodes
(a) Cis electrode (b) Trans electrode 134 Cis-surface 136 Cis-plane
138 Trans-surface 140 Trans-plane 142 Well 142a Well aperture (well
outlet) 144 Sense layer 146 Cavity 148 Sensor aperture 149
Device/Sensor device/Measurement system 150 Integrated circuit 151
Connector 152 Sensing circuit 153 Sensing transistor 154 Sensor
terminal 155 Control circuit 156 Control transistor 158 Control
circuit 160 Compensation circuit 161 Resistor Model 162 Stray
capacitance 164 Flick/control switch 166 Guard switch 168 In-line
amplifier 170 Feedback amplifier 172 Compensation capacitor 174
Select/row-column circuit 176 Row decoder 178 Column readout 180
ADC 182 Control electrode 184 Guard conductor 192 Oxide layers 201
First Schematic Circuit 202 Second Schematic Circuit 203 First
Conductive Element 204 Second Conductive Element 205 Buffer 210
Printed Circuit Board 212 Mechanical Bond 214 Wire Bond 216, 218
Gaskets 222 Solder Bump Connection 224, 226 Flowcells 228 Gasket
230 Sealant 232 Gasket
[0301] While several embodiments of the present disclosure have
been described and illustrated herein, those of ordinary skill in
the art will readily envision a variety of other means and/or
structures for performing the functions and/or obtaining the
results and/or one or more of the advantages described herein, and
each of such variations and/or modifications is deemed to be within
the scope of the present disclosure. More generally, those skilled
in the art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the teachings of the present disclosure
is/are used. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the disclosure described
herein. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within
the scope of the appended claims and equivalents thereto, the
invention may be practiced otherwise than as specifically described
and claimed. The present invention is directed to each individual
feature, system, article, material, and/or method described herein.
In addition, any combination of two or more such features, systems,
articles, materials, and/or methods, if such features, systems,
articles, materials, and/or methods are not mutually inconsistent,
is included within the scope of the present invention.
[0302] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0303] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified unless clearly
indicated to the contrary. Thus, as a non-limiting example, a
reference to "A and/or B," when used in conjunction with open-ended
language such as "comprising" can refer, in one embodiment, to A
without B (optionally including elements other than B); in another
embodiment, to B without A (optionally including elements other
than A); in yet another embodiment, to both A and B (optionally
including other elements); etc.
[0304] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e. "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of." "Consisting essentially of," when used in the
claims, shall have its ordinary meaning as used in the field of
patent law.
[0305] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0306] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," and the like are to
be understood to be open-ended, i.e., to mean including but not
limited to. Only the transitional phrases "consisting of" and
"consisting essentially of" shall be closed or semi-closed
transitional phrases, respectively, as set forth in the United
States Patent Office Manual of Patent Examining Procedures, Section
2111.03.
[0307] Use of ordinal terms such as "first," "second," "third,"
etc., in the claims to modify a claim element does not by itself
connote any priority, precedence, or order of one claim element
over another or the temporal order in which acts of a method are
performed, but are used merely as labels to distinguish one claim
element having a certain name from another element having a same
name (but for use of the ordinal term) to distinguish the claim
elements.
* * * * *